Toxicity screening methods

ABSTRACT

Methods of evaluating an anti-tumor compound based on in vitro assays are disclosed. The methods may determine a potency/efficacy of the anti-tumor compound as well as a toxicity of the anti-tumor compound to non-cancerous cells.

INCORPORATION BY REFERENCE STATEMENT

The following patents and patent applications are hereby expresslyincorporated herein by reference: U.S. Ser. No. 12/570,992, filed Sep.30, 2009; U.S. Ser. No. 11/714,526, filed Mar. 6, 2007, now U.S. Pat.No. 7,615,361, issued Nov. 10, 2009; U.S. Ser. No. 60/779,660, filedMar. 6, 2006; and U.S. Ser. No. 60/743,599, filed Mar. 21, 2006.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

FIELD OF THE INVENTIVE CONCEPT(S)

The present application for patent relates to in vitro methods forpredicting in vivo toxicity of chemical compounds, includingorgan-specific and species-specific toxicity of such chemical compoundsand drug-drug interactions, understanding the relative toxicity of drugcandidates and identifying mechanisms of toxicity.

BACKGROUND

The process of identifying a new drug candidate is long and tedious withmany promising compounds eliminated from development during preclinicaltoxicity testing in animals. One reason for the high number of drop outcompounds during the preclinical phase is the lack of useful toxicitydata early in the discovery program. Many pharmaceutical companies haverecognized this in the last several years. The time and expenseassociated with the drug discovery process has lead to a search forefficiencies that can be realized in the process.

To date, evaluation of in vivo toxicity of a given candidate substanceas a potential drug has involved the use of animal models. Underlyingthe animal tests is the assumption that the effects observed in animalsare applicable and predictive of effects in humans. In general, when thedosage is based on a per unit of body surface area, toxicology data fromanimals is applicable to humans. On the other hand, when the dosage isbased on animal body weight, humans are typically more susceptible totoxicity than the test animals. Nevertheless, the vast majority of drugsare developed to be given on the basis of body weight.

Additionally, the actual numbers of animals used in drug testing aremuch lower than the human population likely to be exposed to the drug ifthe candidate is actually brought to market. For example, a 0.01%incidence of human exposure to a given drug means that approximately25,000 out of 250 million individuals are exposed to a drug. To detectsuch a low incidence in animals would require that 30,000 animals beexposed to the drug. This is clearly an impractical number consideringthe variety of drugs in development at any given time. Consequently,exposure of fewer animals to high doses of candidate substances isdesirable to identify hazards to humans exposed to low doses.

Modern drug development proceeds through a series of stages in which avast library of compounds is gradually narrowed in a series ofsuccessive steps. The use of animals in the initial stages of drugdevelopment, in which the number of compounds still being considered isrelatively large, is an expensive and inefficient method of producingtoxicological data for new drugs especially in light of the fact thatmost chemicals this early in development, ultimately, will not beconsidered drug candidates. Thus, a significant need for alternativetoxicological screening methods exists. Indeed, various approaches totoxicological screening prior to the animal testing stage have beenproposed.

A common approach to solving the toxicology data deficit has been toincorporate in vitro toxicity testing of compounds of interest into thedrug discovery process at a time when new compounds are being identifiedfor potency and efficacy against therapeutic targets. Quality toxicitydata at this early stage permits pharmaceutical chemists to attempt to“design out” toxicity while maintaining efficacy/potency. It has proveddifficult, however, to develop robust in vitro toxicity testing systemsthat provide data that is consistently and reliably predictive of invivo toxicity.

Key issues have been deciding on the type and nature of assays to beutilized and the test system to be employed. There are many biochemicaland molecular assays that claim to assess toxicity in cells grown inculture. However, when only one or even two assays are used over alimited range of exposure concentrations, the probability of falsenegative and false positive data is high. Some of the most commonly usedassays include, but are not limited to, leakage of intracellular markersas determined by lactate dehydrogenase (LDH), glutathione S-transferase(GST), and potassium, and the reduction of tetrazolium dyes such as MTT,XTT, Alamar Blue, and INT. All have been used as indicators of cellinjury. Prior art in vitro toxicity screens typically only involve theuse of one or two endpoints. The resulting data provides a yes/no orlive/dead answer. This minimalist approach to the toxicity-screeningproblem has resulted in little progress towards developing a robustscreening system capable of providing a useful toxicity profile that hasmeaning for predicting similar toxicity in animals. Therefore, thereremains a need in the art for the development of new screening systemsthat provide more useful toxicity information, especially toxicityinformation that can be obtained rapidly and cost-effectively at earlystages of the drug discovery process. A need exists for toxicityscreening systems that do not require the use of animals but thatprovide reliable information on relative toxicity, mechanism oftoxicity, and that effectively predict in vivo toxicity.

The drug discovery process is often under significant time pressures,and any time lost while waiting for toxicity data can prove expensive.Thus, a need also exists for in vitro toxicity screening methods andsystems optimized for providing relevant information relating to invitro toxicity in a relatively short time frame.

In some drug development efforts, it is desirable to evaluate thetoxicity potential for one or more compounds in particular organsystems. Obtaining this information at a late stage in the process canrender significant efforts and expense essentially useless. Thus, a needalso exists for in vitro toxicity screening methods and systems thatprovide relevant information relating to in vivo toxicity in particularorgan systems and functions, such as information relating to the cardiotoxicity potential of a compound.

Some drug discovery efforts implicate toxicity considerations that areof little or no concern in other efforts. For example, most anti-tumordrugs are either cytostatic or cytotoxic. For cytostatic compounds,off-target toxicity is an important consideration considering thedesired result of use of the compound. This consideration is even morecritical for cytotoxic compounds. Thus, a need also exists for in vitrotoxicity screening methods and systems for specific classes of compoundsthat have unique or special considerations, such as compounds beinginvestigated for anti-tumor activity.

An important component of any new drug evaluation is the potential for acompound to exhibit species specific toxicity. For example, in theanimal testing stage of a drug development effort, rodent studies mayshow no adverse signs, while a study in a non-rodent species may showsevere or even lethal toxicity. When this occurs, repeat animal testingmay be required, and significant questions regarding the relevancy ofthe results to human exposure and toxicity can be raised, each of whichcan introduce significant delay and expense into the drug discoveryeffort. Thus, a need also exists for in vitro toxicity screening methodsand systems that provide relevant information relating to potentialspecies-specific toxicity.

Another concern during the drug development process is the potential fordrug-drug interactions in which one drug alters the pharmacokinetics ofa co-administered drug. Having relevant information concerning theability of a compound of interest to be co-administered with otherdrugs, or not to be administered with other drugs, may aid in makingdeterminations as to which compounds should be advanced in the processand which compounds should be halted. Thus, a need also exists for invitro toxicity screening methods and systems that provide relevantinformation relating to the potential for a compound to producedrug-drug interactions in vivo.

It is to such novel toxicity screening systems that the presentlydisclosed and claimed inventive concept(s) is directed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates concentration response analyses for adriamycin inH4IIE (liver cells) and rat cardiomyocytes at 24 hours exposure. Thedual cell model indicates that adriamycin is most toxic to heart.

FIG. 2 illustrates concentration response analyses for Imatinib(GLEEVEC®) in H4IIE cells at 24 hours exposure. This liver cell linereveals mitochondria as the most sensitive target for this drug.

FIGS. 3 and 3A illustrate concentration response analyses for CELEBREX®in H4IIE cells (FIG. 3) and rat cardiomyocytes (FIG. 3A) at 24 hoursexposure. CELEBREX® would have passed as a safe drug in these analyses,and the heart had greater sensitivity than the liver cells at highexposures.

FIGS. 4 and 4A illustrate concentration response analyses for Vioxx inH4IIE cells (FIG. 4A) and rat cardiomyocytes (FIG. 4) at 24 hoursexposure. CELEBREX® and Vioxx produce difference biochemical profiles,suggesting that their effects are not related to their target.

FIG. 4B illustrates lipidosis of H4IIE cells exposed to CELEBREX®,Vioxx, and Amiodarone at 24 hours exposure.

FIGS. 5 and 5A illustrate analyses of the cardiac hypertrophy marker ANPin rat cardiomyocytes exposed to CELEBREX® (FIG. 5) or Vioxx (FIG. 5A).Induction of ANP was measured and normalized with GAPDH with bDNAtechnology.

FIG. 6 contains a graph that illustrates cell specificity for ananti-tumor drug screened therein. Rat primary hepatocytes and normal ratkidney (NRK) cells were not sensitive to the anti-tumor drug, whereasH4IIE cells were highly sensitive to the anti-tumor drug.

FIG. 7 contains a graph that illustrates a toxicity comparison betweenmultiple cell types. Information regarding drug specificity and speciessensitivity can be determined from this type of comparison. SK-MEL28 andC32 are human tumor cell lines; SK-MEL28* refers to data provided by thesponsor. NRK, normal rat kidney cells. HUVEC, human umbilical veinendothelial cells.

FIGS. 8, 8A, 8B, 8C, 8D, 8E, 8F, 8G, and 8H illustrate concentrationresponse analyses for Cisplatin in three rat cell types: H4IIE tumorcells (FIGS. 8, 8A, and 8B), NRK (normal rat kidney cells) (FIGS. 8C,8D, and 8E), and rat primary hepatocytes (FIGS. 8F, 8G, and 8H).

FIGS. 9, 9A, 9B, 9C, 9D, 9E, 9F, 9G, and 9H illustrate concentrationresponse analyses for methotrexate in three rat cell types: H4IIE tumorcells (FIGS. 9, 9A, and 9B), NRK (normal rat kidney cells) (FIGS. 9C,9D, and 9E), and rat primary hepatocytes (FIGS. 9F, 9G, and 9H).

FIGS. 10, 10A, 10B, 10C, 10D, 10E, 10F, 10G, and 10H concentrationresponse analyses for Doxorubicin (Adriamycin) in three rat cell types:H4IIE tumor cells (FIGS. 10, 10A, and 10B), NRK (normal rat kidneycells) (FIGS. 10C, 10D, and 10E), and rat primary hepatocytes (FIGS.10F, 10G, and 10H).

FIGS. 11 and 11A contain bar graphs that illustrate the differentiationof target versus off-target effects (FIG. 11) and cell sensitivity (FIG.11A) for H4IIE, NRK, and primary hepatocyte cells exposed to ananti-tumor drug.

FIG. 12 illustrates a metabolic stability assay of propranolol in ratand human liver microsomes.

FIG. 13 contains a bar graph illustrating the evaluation of drugs withstructural alerts for metabolic activation. APAP, acetaminophen; ABT,1-Aminobenzotriazole, a cytochrome-P450 inhibitor.

FIG. 14 contains total ion chomatograms illustrating a comparison ofpropranolol metabolic profiles in rat and human hepatic microsomes.

FIG. 15 contains selective ion chromatograms illustrating the differentmetabolic profiles in rat and human microsomes.

FIG. 16 illustrates concentration response analyses for a Hep-C proteaseinhibitor, labeled as Compound A, in rat cardiomyocytes following a 1hour exposure.

FIG. 17 illustrates concentration response analyses for Compound A inrat cardiomyocytes following a 3 hour exposure.

FIG. 18 illustrates concentration response analyses for Compound A inrat cardiomyocytes following a 6 hour exposure.

FIG. 19 illustrates concentration response analyses for Compound A inrat cardiomyocytes following a 24 hour exposure.

FIGS. 20A and 20B illustrate concentration response analyses forAdriamycin in rat cardiomyocytes following a 1 hour exposure.

FIGS. 21A and 21B illustrate concentration response analyses forAdriamycin in rat cardiomyocytes following a 3 hour exposure.

FIGS. 22A and 22B illustrate concentration response analyses forAdriamycin in rat cardiomyocytes following a 6 hour exposure.

FIGS. 23A and 23B illustrate concentration response analyses forAdriamycin in rat cardiomyocytes following a 24 hour exposure.

FIGS. 24A and 24B contain bar graphs that illustrate the results of adichlorofluoroscindiacetate (DCFDA) analyses for Compound A (FIG. 24A)and Adriamycin (FIG. 24B) at 1, 3, 6 and 24 hours.

FIGS. 25A, 25B, and 25C contain bar graphs illustrating bDNA analyses ofthe cardiac hypertrophy markers ANP (FIG. 25A), BNP (FIG. 25B), and p53(FIG. 25C) for Compound A in rat cardiomyocytes at 1, 3, 6 and 24 hours.

FIGS. 26A, 26B, and 26C contain bar graphs illustrating bDNA analyses ofthe cardiac hypertrophy markers BAX (FIG. 26A), Bcl2 (FIG. 26B), andiNOS (FIG. 26C) for Compound A in rat cardiomyocytes at 1, 3, 6 and 24hours.

FIGS. 27A, 27B, and 27C contain bar graphs illustrating bDNA analyses ofthe cardiac hypertrophy markers ANP (FIG. 27A), BNP (FIG. 27B), and iNOS(FIG. 27C) for Adriamycin in rat cardiomyocytes at 1, 3, 6 and 24 hours.

FIGS. 28A, 28B, and 28C contain bar graphs illustrating bDNA analyses ofthe cardiac hypertrophy markers BAX (FIG. 28A), Bcl2 (FIG. 28B), and p53(FIG. 28C) for Adriamycin in rat cardiomyocytes at 1, 3, 6 and 24 hours.

FIG. 29 illustrates concentration response analyses for Compound A inH4IIE cells following a 24 hour exposure.

FIG. 30 illustrates concentration response analyses for Adriamycin inH4IIE cells following a 24 hour exposure.

FIG. 31 illustrates concentration response analyses for Idarubicin inrat cardiomyocyte cells following a 24 hour exposure.

FIG. 32 illustrates concentration response analyses for Mitoxantrone inrat cardiomyocyte cells following a 24 hour exposure.

FIG. 33 illustrates concentration response analyses for Daunorubicin inrat cardiomyocyte cells following a 24 hour exposure.

FIG. 34 illustrates concentration response analyses for Pirarubicin inrat cardiomyocyte cells following a 24 hour exposure.

FIG. 35 illustrates concentration response analyses for Epirubicin inrat cardiomyocyte cells following a 24 hour exposure.

FIG. 36 illustrates concentration response analyses for Ritonavir in ratcardiomyocyte cells following a 24 hour exposure.

FIG. 37 illustrates concentration response analyses for Efavirenz in ratcardiomyocyte cells following a 24 hour exposure.

FIG. 38 illustrates concentration response analyses for Lopinavir in ratcardiomyocyte cells following a 24 hour exposure.

FIG. 39 illustrates concentration response analyses for Delavirdine inrat cardiomyocyte cells following a 24 hour exposure.

FIG. 40 illustrates concentration response analyses for Abacavir in ratcardiomyocyte cells following a 24 hour exposure.

FIG. 41 illustrates concentration response analyses for Indinavir in ratcardiomyocyte cells following a 24 hour exposure.

FIG. 42 illustrates concentration response analyses for Nevirapine inrat cardiomyocyte cells following a 24 hour exposure.

FIG. 43 illustrates concentration response analyses for AZT in ratcardiomyocyte cells following a 24 hour exposure.

FIG. 44 illustrates concentration response analyses for Rotenone in ratcardiomyocyte cells following a 24 hour exposure.

FIG. 45 illustrates concentration response analyses for Camptothecin inrat cardiomyocyte cells following a 24 hour exposure.

FIG. 46 illustrates concentration response analyses for Idarubicin inH4IIE cells following a 24 hour exposure.

FIG. 47 illustrates concentration response analyses for Daunorubicin inH4IIE cells following a 24 hour exposure.

FIG. 48 illustrates concentration response analyses for Pirarubicin inH4IIE cells following a 24 hour exposure.

FIG. 49 illustrates concentration response analyses for Doxorubicin inH4IIE cells following a 24 hour exposure.

FIG. 50 illustrates concentration response analyses for Epirubicin inH4IIE cells following a 24 hour exposure.

FIG. 51 illustrates concentration response analyses for Mitoxantrone inH4IIE cells following a 24 hour exposure.

FIG. 52 illustrates concentration response analyses for Efavirenz inH4IIE cells following a 24 hour exposure.

FIG. 53 illustrates concentration response analyses for Ritonavir inH4IIE cells following a 24 hour exposure.

FIG. 54 illustrates concentration response analyses for Delavirdine inH4IIE cells following a 24 hour exposure.

FIG. 55 illustrates concentration response analyses for Lopinavir inH4IIE cells following a 24 hour exposure.

FIG. 56 illustrates concentration response analyses for Abacavir inH4IIE cells following a 24 hour exposure.

FIG. 57 illustrates concentration response analyses for Indinavir inH4IIE cells following a 24 hour exposure.

FIG. 58 illustrates concentration response analyses for Nevirapine inH4IIE cells following a 24 hour exposure.

FIG. 59 illustrates concentration response analyses for AZT in H4IIEcells following a 24 hour exposure.

FIG. 60 illustrates concentration response analyses for Rotenone inH4IIE cells following a 24 hour exposure.

FIG. 61 illustrates concentration response analyses for Camptothecin inH4IIE cells following a 24 hour exposure.

FIG. 62 illustrates concentration response analyses for Rotenone inH4IIE cells following a 6 hour exposure.

FIG. 63 illustrates concentration response analyses of the effects ofRotenone on general cell health and oxidative stress markers in H4IIEcells.

FIGS. 64, 64A, and 64B illustrate concentration response analyses of theeffects of Staurosporine (FIG. 64), Camptothecin (FIG. 64A), andPaclitaxel (FIG. 64B) on general cell health and the apoptosis markerCaspase 3 in H4IIE cells following a 24 hour exposure.

FIG. 65 contains bar graphs illustrating metabolic activation of testcompounds in rat-induced microsomes (top panel) and dog-inducedmicrosomes (bottom panel).

FIG. 66 contains a bar graph illustrating the metabolic stability oftest compounds A-E in rat and dog microsomes.

FIG. 67 contains ion chromatograms illustrating microsomal metabolism ofCompound A in rat and dog.

FIG. 68 contains ion chromatograms illustrating microsomal metabolism ofCompound B in rat and dog.

FIG. 69 contains ion chromatograms illustrating microsomal metabolism ofCompound C in rat and dog.

FIG. 70 contains ion chromatograms illustrating microsomal metabolism ofCompound D in rat and dog.

FIG. 71 contains ion chromatograms illustrating microsomal metabolism ofCompound E in rat and dog.

FIGS. 72A, 72B, and 72C illustrate concentration response analyses ofCompound B in rat primary hepatocytes following a 48 hour exposure(after 24 hour induction with 50 μM PB plus 15 μM BNF).

FIGS. 73A, 73B, and 73C illustrate concentration response analyses ofCompound D in rat primary hepatocytes following a 48 hour exposure(after 24 hour induction with 50 μM PB plus 15 μM BNF).

FIGS. 74A, 74B, and 74C illustrate concentration response analyses ofCompound B in dog primary hepatocytes following a 48 hour exposure(after 24 hour induction with 50 μM PB plus 15 μM BNF).

FIGS. 75A, 75B, and 75C illustrate concentration response analyses ofCompound D in dog primary hepatocytes following a 48 hour exposure(after 24 hour induction with 50 μM PB plus 15 μM BNF).

FIGS. 76A, 76B, 76C, 76D, 76E, 76F, 76G, 76H, 76I, 76J, 76K, 76L, 76M,76N, and 76O illustrate concentration response analyses of Compound Afollowing 6 and 24 hour exposures in a rat hepatoma cell line H4IIE(FIGS. 76A, 76B, 76C, and 76D), rat primary hepatocytes (FIGS. 76E, 76F,76G, 76H, and 76I), and normal rat kidney (NRK) cells (FIGS. 76J, 76K,76L, 76M, 76N, and 76O).

FIGS. 77A, 77B, 77C, 77D, 77E, 77F, 77G, 77H, 77I, and 77J illustrate pHtest analyses of Compound A following 6 and 24 hour exposures in a H4IIEcells (FIGS. 77A and 77B), rat primary hepatocytes (FIGS. 77C, 77D, 77E,and 77F), and NRK cells (FIGS. 77G, 77H, 77I, and 77J).

FIGS. 78A and 78B illustrate concentration response analyses forCamptothecin in NRK cells following a 6 hour exposure.

FIGS. 79A and 79B illustrate concentration response analyses forCamptothecin in NRK cells following a 24 hour exposure.

FIGS. 80A and 80B illustrate concentration response analyses forRotenone in NRK cells following a 6 hour exposure.

FIGS. 81A and 81B illustrate concentration response analyses forRotenone in NRK cells following a 24 hour exposure.

FIG. 82 contains a graph that illustrates cell specificity for ananti-tumor drug screened therein. Rat primary hepatocytes and normal ratkidney (NRK) cells were not sensitive to the anti-tumor drug, whereasH4IIE cells were highly sensitive to the anti-tumor drug.

FIG. 83 contains a bar graph that illustrates a toxicity comparisonbetween multiple cell lines. SK-MEL28 and C32 are human tumor celllines; SK-MEL28* refers to data provided by the sponsor. NRK, normal ratkidney cells. NHEM, Normal Human Epidermal Melanocyte. HUVEC, humanumbilical vein endothelial cells.

FIGS. 84A, 84B, 84C, and 84D contain concentration response analyses forCompound A in SK-MEL28 tumor cells following a 24 hour exposure, and MTTcombined low/high exposures.

FIGS. 85A, 85B, 85C, 85D, 85E, and 85F illustrate concentration responseanalyses for Compound A (low dose exposure) in SK-MEL28 cells following24 and 72 hour exposures.

FIGS. 86A, 86B, and 86C illustrate concentration response analyses forCompound A in human hepatocytes following a 24 hour exposure.

FIGS. 87A, 87B, and 87C illustrate concentration response analyses forCompound A in HUVEC (human umbilical vein endothelial cells) following a24 hour exposure.

FIGS. 88A, 88B, and 88C illustrate concentration response analyses forCompound A in C32 tumor cells following a 24 hour exposure.

FIGS. 89A, 89B, 89C, 89D, 89E, and 89F illustrate concentration responseanalyses for Compound A in C32 cells (low exposure) following a 24 hourexposure, and combined low/high exposures.

FIGS. 90A, 90B, and 90C illustrate concentration response analyses forCompound A in NHEM cells following a 24 hour exposure.

FIGS. 91A, 91B, and 91C illustrate concentration response analyses forCamptothecin in SK-MEL28 cells following a 24 hour exposure.

FIGS. 92A, 92B, 92C, 92D, 92E, and 92F illustrate concentration responseanalyses for Camptothecin in SK-MEL28 cells following 24 and 72 hourexposures.

FIGS. 93A, 93B, and 93C illustrate concentration response analyses forCamptothecin in human hepatocytes following a 24 hour exposure.

FIGS. 94A, 94B, and 94C illustrate concentration response analyses forCamptothecin in human umbilical vein endothelial cells (HUVEC) followinga 24 hour exposure.

FIGS. 95A, 95B, and 95C illustrate concentration response analyses forCamptothecin in C32 cells following a 24 hour exposure.

FIGS. 96A, 96B, and 96C illustrate concentration response analyses forCamptothecin in NHEM cells following a 24 hour exposure.

FIGS. 97A, 97B, and 97C illustrate concentration response analyses forRotenone in SK-MEL28 cells following a 24 hour exposure.

FIGS. 98A, 98B, 98C, 98D, 98E, and 98F illustrate concentration responseanalyses for Rotenone in SK-MEL28 cells following 24 and 72 hourexposures.

FIGS. 99A, 99B, and 99C illustrate concentration response analyses forRotenone in human hepatocytes following a 24 hour exposure.

FIGS. 100A, 100B, and 100C illustrate concentration response analysesfor Rotenone in human umbilical vein endothelial cells (HUVEC) followinga 24 hour exposure.

FIGS. 101A, 101B, and 101C illustrate concentration response analysesfor Rotenone in C32 cells following a 24 hour exposure.

FIGS. 102A, 102B, and 102C illustrate concentration response analysesfor Rotenone in NHEM cells following a 24 hour exposure.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following detailed description and appendices describe andillustrate various exemplary embodiments. The description and drawingsserve to enable one skilled in the art to make and use the presentlydisclosed and claimed inventive concept(s), and are not intended tolimit the scope of the presently disclosed and claimed inventiveconcept(s) in any manner.

Relevant background information is available in U.S. Pat. No. 6,998,249issued to McKim and Cockerell on Feb. 14, 2006 and entitled “TOXICITYSCREENING METHOD”, the contents of which are expressly incorporated intothis disclosure in their entirety.

Before explaining at least one embodiment of the presently disclosed andclaimed inventive concept(s) in detail by way of exemplary drawings,experimentation, results, and laboratory procedures, it is to beunderstood that the presently disclosed and claimed inventive concept(s)is not limited in its application to the details of construction and thearrangement of the components set forth in the following description orillustrated in the drawings, experimentation and/or results. Thepresently disclosed and claimed inventive concept(s) is capable of otherembodiments or of being practiced or carried out in various ways. Assuch, the language used herein is intended to be given the broadestpossible scope and meaning; and the embodiments are meant to beexemplary—not exhaustive. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

Unless otherwise defined herein, scientific and technical terms used inconnection with the presently disclosed and claimed inventive concept(s)shall have the meanings that are commonly understood by those ofordinary skill in the art. Further, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Generally, nomenclatures utilized in connectionwith, and techniques of, cell and tissue culture, molecular biology, andprotein and oligo- or polynucleotide chemistry and hybridizationdescribed herein are those well known and commonly used in the art.Standard techniques are used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual (2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) and Coligan et al. Current Protocols in Immunology(Current Protocols, Wiley Interscience (1994)), which are incorporatedherein by reference. The nomenclatures utilized in connection with, andthe laboratory procedures and techniques of, analytical chemistry,synthetic organic chemistry, and medicinal and pharmaceutical chemistrydescribed herein are those well known and commonly used in the art.Standard techniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

Presently, it takes between three and five years to bring newpotentially therapeutic compounds from the early discovery process topreclinical development (in vivo animal testing). The toxicity data forsuch compounds is generally not available until the preclinical animaltoxicity tests are performed. Numerous drugs reach this stage ofdevelopment only to be discarded from further development due to toxicliability. Such failures represent a tremendous loss in companyresources. Understandably, a technique that could predict the toxicityof these multitudes of compounds that arrive at or near the initialsynthesis stage of drug discovery would have an enormous impact on theefficiency with which new drugs are identified by eliminating, early inthe drug discovery process, compounds that have an unfavorable toxicityprofile. An immediate expected benefit of more powerful, early stage invitro toxicity testing is the reduction of the number of these three tofive year drug discovery cycles that result in failures, and thus reducethe average number of cycles required to develop successful newtherapeutics. Related benefits include reduced costs for drugdevelopment and more rapid availability of new pharmaceuticals to themedical community. Reduced failure rates may lengthen the portion of apatent's term that valuable compounds enjoy commercial exploitation.

In addition, in the animal testing stage of a drug development effort,one animal species may show no adverse effects upon administration of adrug, whereas a second animal species may exhibit severe or even lethaltoxicity. When this occurs, repeat animal testing may be required, andsignificant questions regarding the relevancy of the results to humanexposure and toxicity can be raised. Therefore, the ability to identifythese species-specific toxicity differences in vitro would be of greatvalue.

The presently disclosed and claimed inventive concept(s) providesmethods for prioritizing new chemical entities within a class forfurther development, identifying mechanisms of toxicity andorgan-specific and species-specific toxicities and for estimating invivo toxicity early in the drug development and discovery process. Assuch, these methods can be used to prioritize large numbers of newcompounds for further drug development. In addition, the methods greatlyincrease the probability that an identified agent will be successful inpreclinical toxicity testing. The adaptability of these in vitro methodsfor high-throughput analysis makes them an economical and cost-effectiveaddition to a drug discovery program.

In particular, the presently disclosed and claimed inventive concept(s)provides a cluster analysis in which two or more different biochemicalendpoints are evaluated in order to predict the in vivo toxicityconcentration of a given compound, prioritize compounds based onrelative toxicity, and identify mechanisms of toxicity. In certainembodiments, these assays measure changes in specific biochemicalprocesses, which are essential for normal cellular functions, followinga 24-hour exposure to a broad range of concentrations of the compound.In certain other embodiments, these assays may be measured followingboth a 6-hour exposure and a 24-hour exposure to a broad range ofconcentrations of the compound.

The toxicity cluster analyses of the presently disclosed and claimedinventive concept(s) allow the determination of appropriate informationrelating to changes occurring in specific cellular processes. Thisinformation in turn is used to obtain a more complete profile ofcellular injury and/or cytotoxicity. Further, the analyses describedherein may be utilized to identify specific types of toxicity, i.e.,toxicity to certain cell types such as but not limited to, cardiaccells; toxicity due to drug-drug interactions; species-specifictoxicity, and the like.

I. Cluster Analysis Toxicity Screening

In the presently disclosed and claimed inventive concept(s), clusteranalysis toxicity screening is presented as a method of predicting thein vivo toxicity of a given compound. In particular aspects, theseassays will involve culturing cells in culture medium that comprises aplurality of concentrations of the chemical compound; measuring aplurality of cell health indicators of the cell in response to culturingin at least three concentrations of the chemical compound and predictingTC₅₀ and a toxic concentration (C_(tox)) of the chemical compound fromsuch measurements. The various embodiments involved in conducting suchassays are described in further detail below.

Assay Format

In certain embodiments, the CATS technique will be used to prioritizeand identify compounds that will be of a potential therapeutic value.The inventors have discovered that analyzing multiple endpoints yieldssignificant information regarding the toxicity of a given compound.

In certain embodiments, the presently disclosed and claimed inventiveconcept(s) concerns a method for identifying such compounds. It iscontemplated that this screening technique will prove useful in thegeneral prioritization and identification of compounds that will serveas lead therapeutic compounds for drug development. The presentlydisclosed and claimed inventive concept(s) will be a useful addition tolaboratory analyses directed at identifying new and useful compounds forthe intervention of a variety of diseases and disorders including, butnot limited to, Alzheimer's disease, other disorders and diseases of thecentral nervous system, metabolic disorders and diseases, cancers,diabetes, depression, immunodeficiency diseases and disorders,immunological diseases and disorders, autoimmune diseases and disorders,gastrointestinal diseases and disorders, cardiovascular diseases anddisorders, inflammatory diseases and disorders, and infectious diseases,such as a microbial, viral or fungal infections.

In specific embodiments, the presently disclosed and claimed inventiveconcept(s) is directed to a method for determining the in vivocytotoxicity of a candidate substance by employing a method includinggenerally: a) culturing cells in culture medium that comprises aplurality of concentrations of said chemical compound; b) measuring afirst indicator of cell health at four or more concentrations of saidchemical compound; c) measuring a second indicator of cell health atfour or more concentrations of said chemical compound; d) measuring athird indicator of cell health at four or more concentrations of saidchemical compound; and e) predicting a toxic concentration (C_(tox)) ofsaid chemical compound from the measurements of steps (b), (c) and (d).

In certain aspects, the method may further involve predicting a TC₅₀ ofsaid chemical compound from the measurements of steps (b), (c) and (d).For any particular assay, the TC₅₀ represents the concentration of acompound which causes fifty percent of a maximal toxic response in theassay. As described in greater detail below, when CATS is run undercertain conditions, TC₅₀ can be selected as a predicted C_(tox).

The foregoing method requires preparing cell cultures. Such a cell maybe a primary cell in culture or it may be a cell line. The cells may beobtained from any mammalian source that is amenable to primary cultureand/or adaptation into cell lines. In lieu of generating cell lines fromanimals, such cell lines may be obtained from, for example, AmericanType Culture Collection, (ATCC, Rockville, Md.), or any other Budapesttreaty or other biological depository. The cells used in the assays maybe from an animal source or may be recombinant cells tailored to expressa particular characteristic of, for example, a particular disorder forwhich the drug development is being considered. In one embodiment, thecells are derived from tissue obtained from humans or other primates,rats, mice, rabbits, sheep, dogs and the like. Techniques employed inmammalian primary cell culture and cell line cultures are well known tothose of skill in that art. Indeed, in the case of commerciallyavailable cell lines, such cell lines are generally sold accompanied byspecific directions of growth, media and conditions that are preferredfor that given cell line.

The presently disclosed and claimed inventive concept(s) predicts thecytotoxicity of a given compound by measuring two or more indicators ofcell health in a given cell. The cell chosen for such an endeavor willdepend on the putative site of in vivo toxicity to be determined. Forexample, the liver is a particularly prevalent site of in vivo drugtoxicity. Thus, the use of liver cells (either primary or cell linesderived from liver cells) in the assays described herein is specificallycontemplated. In certain embodiments, the inventors have found that theH4IIE cell line (ATCC #CRL-1548) is an excellent candidate forpredicting the cytotoxic effects of compounds on the general health ofhepatic cells. In addition, because the H4IIE cell line is aproliferating cell population, the system will be useful in identifyingcompounds that adversely affect other proliferating cell types such ashematopoietic cells. Such cells can be used to identify chemotherapeuticagents that have extremely low hepatotoxicity but high toxicity toproliferating cells. (See Example 5 of U.S. Pat. No. 6,998,249, issuedto McKim et al. on Feb. 14, 2006, the contents of which have beenincorporated herein previously).

While the H4IIE cell line is described herein as a particularlycontemplated cell line, it should be understood that any mammalianprimary hepatic cell or hepatic cell line will be useful in thepresently disclosed and claimed inventive concept(s). In certainembodiments, the cell is a rat hepatic cell line. In addition to H4IIE,other rat cell lines contemplated for use in the presently disclosed andclaimed inventive concept(s) include, but are not limited to MH1C1 (ATCCCCL144), clone 9 (ATCC CRL-1439), BRL 3A (ATCC CRL-1442), H4TG (ATCCCRL-1578), H4IIEC3 (ATCC CRL-166), McA-RH7777 (ATCC CRL-1601) McA-RH8994(ATCC CRL-1602), N1-S1 Fudr (ATCC CRL-1603) and N1-S1 (ATCC CRL-1604).

In other embodiments, the cell is a human liver cell. A human hepaticcell line acceptable for use in the methods described by the presentlydisclosed and claimed inventive concept(s) is HepG2 (ATCC HB-8065).Additionally, other exemplary human hepatic cell lines that may beuseful in the presently disclosed and claimed inventive concept(s)include but are not limited to C3A (ATCC CRL-10741), DMS (ATCCCRL-2064), SNU-398 (ATCC CRL-2233), SNU-449 (ATCC CRL-2234), SNU-182(ATCC CRL-2235), SNU-475 (ATCC CRL2236), SNU-387 (ATCC CRL-2237),SNU-423 (ATCC CRL-2238), NCI-H630 (ATCC CRL-5833), NCI-H1755 (ATCCCRL-5892), PLC/PRF/5 (ATCC CRL8024), Hep3B (HB-8064) and HTB-52 (ATCCHTB-52).

While the above cells will be useful indicators of hepatic celltoxicity, the presently disclosed and claimed inventive concept(s) maybe employed to determine, monitor or otherwise predict cytotoxicity in avariety of tissue types. It should be understood that the in vivo sitesof cellular toxicity that those of skill in the art will want to monitorwill include the in vivo sites of action of the particular test compoundas well as sites remote from the site of action of the test compound.Therefore, cell lines that may be used in assays will include cell linesderived from other common sites of in vivo cytotoxicity such as thekidney, heart and pancreas. While these tissues, along with the liver,may be the primary tissues that one would select to monitorcytotoxicity, it should be understood that the assays of the presentlydisclosed and claimed inventive concept(s) may be employed to predictthe cytotoxic effects of a test compound on cells derived from brain,nerve, skin, lung, spleen, endometrial, stomach and breast tissue, aswell as stem cells and hematopoietic cells. Use of hematopoietic cellsor “stem” cells or cell lines derived therefrom in cytotoxicity assaysis particularly contemplated.

For example, one embodiment of the presently disclosed and claimedinventive concept(s) is directed to a method of determining a level ofcardiac toxicity of a chemical compound. For such analyses, primarycardiac cells would be utilized. In particular, freshly isolatedcardiomyocytes from 7-day old rats may be utilized in accordance withthe presently disclosed and claimed inventive concept(s).

In another embodiment, the presently disclosed and claimed inventiveconcept(s) may include performing such methods with more than one celltype. For example, to analyze the toxicity of an anti-tumor compound, itwould be beneficial to examine the effects of the compound on differentcell types, i.e., cancer-derived proliferating cells, proliferatingcells derived from normal tissue, and non-proliferating cells derivedfrom normal tissue. The use of these different cell types allows for thedifferentiation between target versus off target effects of theanti-tumor compound.

Alternatively, the same cell type from two or more different mammalianspecies may be utilized in accordance with the presently disclosed andclaimed inventive concept(s). The use of cells from different speciesallows for the identification of potential species specific toxicity ofa compound.

In particular embodiments, the cells are seeded in multiwell (e.g.,96-well) plates and allowed to reach log phase growth. In H4IIE cells,this growth period is approximately 48 hours. Preferred media and cellculture conditions for this cell-line are detailed in the Examples.

Once the cell cultures are thus established, various concentrations ofthe compound being tested are added to the media and the cells areallowed to grow exposed to the various concentrations for 24 hours.While the 24 hour exposure period is described, it should be noted thatthis is merely an exemplary time of exposure and testing the specificcompounds for longer or shorter periods of time is contemplated to bewithin the scope of the presently disclosed and claimed inventiveconcept(s). As such it is contemplated that the cells may be exposed for6, 12, 24, 36, 48 or more hours. Increased culture times may sometimesreveal additional cytotoxicity information, at the cost of slowing downthe screening process.

Furthermore, the cells may be exposed to the test compound at any givenphase in the growth cycle. For example, in some embodiments, it may bedesirable to contact the cells with the compound at the same time as anew cell culture is initiated. Alternatively, it may be desirable to addthe compound when the cells have reached confluent growth or arc in loggrowth phase. Determining the particular growth phase cells are in isachieved through methods well known to those of skill in the art.

The varying concentrations of the given test compound are selected withthe goal of including some concentrations at which no toxic effect isobserved and also at least two or more higher concentrations at which atoxic effect is observed. A further consideration is to run the assaysat concentrations of a compound that can be achieved in vivo. Forexample, assaying several concentrations within the range from 0micromolar to about 300 micromolar is commonly useful to achieve thesegoals. It will be possible or even desirable to conduct certain of theseassays at concentrations higher than 300 micromolar, such as, forexample, 350 micromolar, 400 micromolar, 450 micromolar, 500 micromolar,600 micromolar, 700 micromolar, 800 micromolar, 900 micromolar, or evenat millimolar concentrations. The estimated therapeutically effectiveconcentration of a compound provides initial guidance as to upper rangesof concentrations to test. Additionally, as explained in greater detailbelow, CATS analysis may further include assaying a range ofconcentrations that includes at least two concentrations at whichcytotoxicity is observable in an assay. It has been found that assayinga range of concentrations as high as 300 micromolar often satisfies thiscriterion.

In an exemplary set of assays, the test compound concentration rangeunder which the CATS is conducted comprises dosing solutions which yieldfinal growth media concentration of 0.05 micromolar, 0.1 micromolar, 1.0micromolar, 5.0 micromolar, 10.0 micromolar, 20.0 micromolar, 50.0micromolar, 100 micromolar, and 300 micromolar of the compound inculture media. As mentioned, these are exemplary ranges, and it isenvisioned that any given assay will be run in at least two differentconcentrations, and the concentration dosing may comprise, for example,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more concentrations of thecompound being tested. Such concentrations may yield, for example, amedia concentration of 0.05 micromolar, 0.1 micromolar, 0.5 micromolar,1.0 micromolar, 2.0 micromolar, 3.0 micromolar, 4.0 micromolar, 5.0micromolar, 10.0 micromolar, 15.0 micromolar, 20.0 micromolar, 25.0micromolar, 30.0 micromolar, 35.0 micromolar, 40.0 micromolar, 45.0micromolar, 50.0 micromolar, 55.0 micromolar, 60.0 micromolar, 65.0micromolar, 70.0 micromolar, 75.0 micromolar, 80.0 micromolar, 85.0micromolar, 90.0 micromolar, 95.0 micromolar, 80.0 micromolar, 110.0micromolar, 120.0 micromolar, 130.0 micromolar, 140.0 micromolar, 150.0micromolar, 160.0 micromolar, 170.0 micromolar, 180.0 micromolar, 190.0micromolar, 200.0 micromolar, 210.0 micromolar, 220.0 micromolar, 230.0micromolar, 240.0 micromolar, 250.0 micromolar, 260.0 micromolar, 270.0micromolar, 280.0 micromolar, 290.0 micromolar, and 300 micromolar inculture media. It will be apparent that a cost-benefit balancing existsin which the testing of more concentrations over the desired rangeprovides additional information, but at additional cost, due to theincreased number of cell cultures, assay reagents, and time required. Inone embodiment, ten different concentrations over the range of 0micromolar to 300 micromolar are screened.

Typically, the various assays described in the present specification mayemploy cells seeded in 96 well plates or 384 cell plates. The cells arethen exposed to the test compounds over a concentration range, forexample, 0-300 micromolar. The cells are incubated in theseconcentrations for a given period of, for example, 6 and/or 24 hours.Subsequent to the incubation, the assays of the cluster are performedfor each test compound. In one embodiment, all the assays are performedat the same time such that a complete set of data are generated undersimilar conditions of culture, time and handling. However, it may bethat the assays are performed in batches within a few days of eachother.

In specific embodiments, the indicators of cell health and viabilityinclude but are not limited to, indicators of cellular replication,mitochondrial function, energy balance, membrane integrity and cellmortality. In other embodiments, the indicators of cell health andviability further include indicators of oxidative stress, metabolicactivation, metabolic stability, enzyme induction, enzyme inhibition,and interaction with cell membrane transporters.

The compounds to be tested may include fragments or parts ofnaturally-occurring compounds or may be derived from previously knowncompounds through a rational drug design scheme. It is proposed thatcompounds isolated from natural sources, such as animals, bacteria,fungi, plant sources, including leaves and bark, and marine samples maybe assayed as candidates for the presence of potentially usefulpharmaceutical compounds. Alternatively, pharmaceutical compounds to bescreened for toxicity could also be synthesized (i.e., man-madecompounds).

The types of compounds being monitored may be antiviral compounds,antibiotics, anti-inflammatory compounds, antidepressants, analgesics,antihistamines, diuretic, antihypertensive compounds, antiarrythmiadrugs, chemotherapeutic compounds for the treatment of cancer,antimicrobial compounds, among others.

Regardless of the source or type of the compound to be tested forcytotoxicity, it may be necessary to monitor the biological activity ofthe compounds to provide an indication of the therapeutic efficacy of aparticular compound or group of compounds. Of course, such assays willdepend on the particular therapeutic indication being tested. Exemplaryindications include efficacy against Alzheimer's disease, cancer,diabetes, depression, immunodeficiency, autoimmune disease,gastrointestinal disorder, cardiovascular disease, inflammatory diseaseand the like.

Cluster Analyses Assays

The use of multiple assays to develop a toxicity profile for new drugsproves to be a very powerful tool for accurately assessing the effectsof a compound in a living system.

Selective assays used in the clusters of the presently disclosed andclaimed inventive concept(s) provide key information pertaining to thetoxicity profile of a given compound. The assays may be performed suchthat information regarding the various parameters is obtained at thesame time during the drug development phase of drug discovery as opposedto performing the assays at different times during the drug developmentscheme. In one embodiment, the assays are performed in a batch all atthe same time. In other aspects, it may be useful to perform the assayson cell cultures all generated at the same time from an initial cellline.

Modules may be designed in which a cluster of assays address a specificconcern. Thus, in order to monitor the effect of a specific compound onthe general health of a cell, monitoring membrane integrity,mitogenesis, mitochondrial function and energy balance will beparticularly useful. The specific assay employed for any of theseendpoints is not considered to be limiting. Thus, any assay thatprovides an indication of membrane integrity may be combined with anyassay that is predictive of mitogenesis (cell replication) along withany assay that is an indicator of mitochondrial function and energybalance.

In addition to a module for determining the general cell health, othermodules of interest would include those that are directed to determiningfor example, oxidative stress, cell cycle parameters, acute inflammatoryresponse, apoptosis, endocrine responses and interaction with cellmembrane transporters such as Pgp.

In a module that determines oxidative stress, production of reactiveoxygen species (ROS), reactive nitrogen species (RNS), or lipidperoxidation may be monitored. Exemplary assays to be employed in thecluster may involve monitoring endpoints that include but are notlimited to glutathione/glutathione disulfide (GSH/GSSG),dichlorofluoroscindiacetate (DCFDA), lipid peroxidation, 8-isoprostane,8-oxy guanine (8-oxy G) DNA adducts, thiobarbituric acid (TBARS), andmalondialdehyde (MDA).

Modules designed to monitor cell cycle may include determining theeffect on the presence or level of any given cell cycle indicatorincluding but not limited to p53, p21, TGFβ, CDK1, PCNA, telomerase,nitric oxide, and inducible nitric oxide synthase (iNOS). Again anyparticular assay may be employed to determine the level or amount of anygiven cell cycle indicator.

Modules to monitor apoptosis may include any assays described herein orotherwise known in the art. One example of such an assay is a caspase-3assay; however, the presently disclosed and claimed inventive concept(s)is to be understood to not be limited to the use of such assay, and anyapoptosis assay may be substituted therefor in accordance with thepresently disclosed and claimed inventive concept(s).

In a module designed to determine interactions with cell membranetransporter, an exemplary assay to be employed in the cluster mayinvolve measuring a chemical compound's interaction with P-glycoprotein(Pgp). Pgp is a well characterized human ABC-transporter of the MDR/TAPsubfamily. It is extensively distributed and expressed in normal cellssuch as those lining the intestine, liver cells, renal proximal tubularcells, and capillary endothelial cells comprising the blood brainbarrier. Pgp is an ATP-dependent efflux pump with broad substratespecificity that likely evolved as a defense mechanism against harmfulsubstances. Pgp transports various substrates across the cell membrane,thus allowing for the regulation of the distribution and bioavailabilityof drugs.

As stated above the specific assay to monitor any of the givenparameters is not considered crucial so long as that assay is consideredby those of skill in the art to provide an appropriate indication of theparticular biochemical or molecular biological endpoint to bedetermined, such as information about mitochondrial function, energybalance, membrane integrity, cell replication, and the like. Thefollowing sections provide exemplary assays that may be used in thecontext of the presently disclosed and claimed inventive concept(s).This is not intended to be an exhaustive treatise on the description ofthese assays but rather is to be a guidance as to the type of assaysthat are available to those of skill in the art.

Compounds that produce direct effects on the cells typically altermitochondrial function, by either up- or down regulating oxidativerespiration. This means that cellular energy in the form of ATP may bealtered. Mitochondrial function can be used as an indicator ofcytotoxicity and cell proliferation. Healthy mitochondria catalyze thereduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) to a blue or purple formazan compound. The relativelyinsoluble formazan blue is extracted into isopropanol and the absorbanceof the extract measured. A high absorbance value indicates viable cellsand functional mitochondria. Conversely, a decrease in the intensity ofcolor suggests either a loss of cells, or direct toxic effects on themitochondria. The MTT assay is well known to those of skill in the artand has been described in, for example, the MTT mitochondrial dye assayis described in Mosmann, J Immunol. Methods 65, 55-63, 1983 and inDenizot et al., J Immunol. Methods. 89, 271-277, 1986. A similar assaythat monitors XTT mitochondrial dye is described by Roehm et al., J.Immunol. Methods, 142, 257-265, 1991. In addition, those of skill in theart also may determine mitochondrial function by performing, forexample, an Alamar Blue assay [Goegan et al., Toxicol. In vitro 9,257-266. 1995], a Rhodamine 123 assay, or a cytochrome C oxidase assay.

ATP provides the primary energy source for many cellular processes andis required to sustain cell and tissue viability. Intracellular levelsof ATP decrease rapidly during necrosis or apoptosis. Therefore, changesin the cellular concentration of ATP can be used as a general indicatorof cell health. When normalized on a per cell basis, ATP can provideinformation on the energy status of the cell and may provide a marker toassess early changes in glycolytic or mitochondrial function. Assaysthat allow a determination of ADP/ATP energy balance are well known inthe art (Kangas et al., Med Biol, 62, 338-343, 1984).

Measurements of α-GST leakage from cultured cells into the media can beused to assess membrane integrity. This assay is specific for the alphaform of GST that exists at high concentrations in the cytosol ofhepatocytes. An ELISA kit purchased from Biotrin Inc. was used tomeasure GST. GST leakage assays, have been described in the literature,for example, Redick et al., J Biol. Chem. 257, 15200-15203. Oberley etal., Toxicol. Appl. Pharmacol. 131, 94-107, 1995; Feinfeld, J Clin ChemClin Biochem. 24, 529-532, 1986.

Other assays for determining membrane integrity include, but are notlimited to, assays that determine lactate dehydrogenase activity,aspartyl aminotransferase, alanine aminotransferase, isocitratedehydrogenase, sorbitol dehydrogenase, glutamate dehydrogenase,ornithine carbamyl transferase, γ-glutamyl transferase, and alkalinephosphatase.

The ability of cells to divide requires coordinated signaling between avast array of intracellular receptors. Cell replication or “mitogenesis”requires the cells to be functioning at optimum. A change in the abilityto replicate is therefore an indication of stress or abnormal function.An exemplary assay that will allow the determination of cell replicationis the CYQUANT® assay system from Invitrogen, Molecular Probes(Carlsbad, Calif.). Additional assays that may be used to provide anindication of mitogenesis may include, but are not limited to,monitoring ³H-thymidine incorporation and a BrdU incorporation assay. Inaddition, mitogenesis may be monitored by determining the function,presence or absence of a component that controls cell cycle. Exemplarycomponents will be well known to those of skill in the art and include,but are not limited to, p53, p21, TGF-β, CDK1, PCNA and the like.

Certain of the assays performed as part of the CATS analysis willinvolve measuring components of the media whereas others will involvemeasuring cell number or parameters from the cells or cell lysates. TheCATS analysis advantageously involves selecting some assays that can usemedia and others that can use cells from a single well.

Predicting In Vivo Toxicity of a Compound from In Vitro Analyses

Once all data for a given cluster of assays are received, the data areanalyzed to obtain a detailed profile of the compound's toxicity. Forexample, most conveniently, the data are collated over a dose responserange on a single graph. In such an embodiment, the measurementevaluated for each parameter (i.e., each indicator of cell health) atany given concentration is plotted as a percentage of a controlmeasurement obtained in the absence of the compound. However, it shouldbe noted that the data need not be plotted on a single graph, so long asall the parameters are analyzed collectively to yield detailedinformation of the effects of the concentration of the compound on thedifferent parameters to yield an overall toxicity profile. As set forthbelow, this overall toxicity profile will facilitate a determination ofa plasma concentration C_(tox) that is predicted to be toxic in vivo.C_(tox) represents an estimate of the sustained plasma concentration invivo that would result in toxicity, such as hepatotoxicity orhematopoietic toxicity.

A fundamental premise in the field of toxicology is that all compoundsare poisons, and that it is the dose of the compound that determines abeneficial/therapeutic effect versus a toxic effect. Dose is affected bytime of exposure, dosing regimens, pharmacokinetic parameters such asabsorption, metabolism and elimination, by difference between speciesbeing treated, and by route of administration. All these factorsinfluence the plasma concentration of a drug and its duration ofexposure. Thus, in principle, in vitro screens need only account formetabolism and time of exposure. In theory, an increased exposure timeshould shift the dose response curve to the left (e.g., TC₅₀ is lower orthe compound appears more toxic over longer exposure times). Thesefactors all have been considered in the selection of C_(tox) in the CATSassay.

For example, in vitro time course experiments utilizing H4IIE cells wereconducted to evaluate the change in the dose-response curves forchloramphenical and ketoconazole over a 72 hour exposure period. Thedata indicated that the largest shift in TC₅₀ values occurred between 24and 48 hours and that extended exposures (72 hours) had little or noeffect on the toxicity profile. From these data it was determined thatthe NOEL of the 24-hour exposure correlated well with in vivo toxicityand provided acceptable estimates of the plasma concentration in vivothat would result in toxicity. When compared to in vivo animal studies,the 24-hour TC₅₀ concentration for the most sensitive toxicity exceededthe concentration at which toxicity occurred. However, the NOEL of the24 hour period also correlated with the 72-hour TC₅₀ concentration forthe more sensitive assays, and thus the 72-hour TC₅₀ concentration alsoprovided acceptable estimates of the plasma concentration in vivo thatwould result in toxicity.

Studies such as these indicate that a preferred concentration forsetting C_(tox) is the highest concentration at which there is noobserved effect on any of the indicators being measured in the clusteranalysis, especially a 24-hour cluster analysis. The TC₅₀ concentrationin the most sensitive of toxicity assays in a 72 hour cluster analysishas been observed to correlate with the 24 hour NOEL/C_(tox), and thusrepresents another datapoint in the CATS analysis that works as anestimate of the sustained plasma concentration in vivo that would resultin toxicity. It will be apparent that 24 hour assays are moretime-effective, and consequently, the 24 hour NOEL/C_(tox) represents apreferred data point to select as C_(tox) in a CATS assay. It will alsobe apparent that, with further time studies, it may be possible toselect an equally suitable C_(tox) at other CATS assay time points(e.g., between 24 and 72 hours, or less than 24 hours).

In certain embodiments, the predicting comprises performingconcentration response analyses of measurements from at least threeseparate assays that are employed in the cluster analysis. For example,in the cell health cluster, such predicting will involve monitoring theconcentration response effect of the compound on a first healthindicator which monitors cellular replication, a second cell healthindicator which monitors mitochondrial function, and a third cell healthindicator which monitors membrane integrity. Of course, it is understoodthat fourth, fifth, sixth or more cell health indicators also may beemployed. From these concentration response analyses, the highestconcentration of the chemical compound at which a measurable toxiceffect of the chemical compound is not observable, i.e., NOEL, isdetermined and the C_(tox) is identified as the concentration thatcorrelates to the NOEL. In choosing the concentrations of the compoundfor analysis, one of skill in the art should devise a dose responseregimen which is selected to provide an indication of cell health atconcentrations of at least two concentration values higher than theC_(tox) concentration.

In the specific embodiments, the results of the analyses are depicted ona single graph on which the values are presented relative to control.The term “relative to control” means that the measurements in thepresence of a given concentration of the compound are compared to asimilar assay performed in the absence of the compound. The measurementin the absence of the compound is presented as the 100% measurement. Theeffect of the compound is thus determined as a raw figure which is thenadjusted relative to that measurement that is determined in the absenceof the compound.

In certain instances, there may be enough biological activityinformation generated for a compound or series of compounds fromefficacy/activity experiments to predict a plasma concentration inhumans that will be required to see a therapeutic effect. Even wheresuch a prediction is premature, there may at least be some activity dataindicating concentration of a compound or series of compounds needed toachieve a biological effect that correlates with a desired therapeuticactivity. In such instances, it becomes possible to use the in vitrodata from CATS analysis to estimate a therapeutic index (TI). TI for adrug is calculated by dividing the toxic concentration (conventionally aTC₅₀ value) by the beneficial therapeutic concentration. Thus, thelarger the TI number, the safer the drug. For example, for a compoundwhich has a TC₅₀ value greater than 100 micromolar, an estimated C_(tox)value of 50 micromolar and an estimated therapeutic concentration of 0.2micromolar, a TI of 500 is obtained. If the estimated C_(tox) is used asthe toxic concentration, then a TI of 250 is obtained. This wouldrepresent a safe drug at least in terms of liver toxicity. A TI that isat least 10 is preferred, and a TI of 100 is particularly preferred. Ofcourse, values higher than 100 will be indicative of the drug beingespecially safe and would be most preferred.

Thus, in one embodiment of the presently disclosed and claimed inventiveconcept(s) useful for prioritizing candidate therapeutic agents, oneperforms an in vitro activity assay to determine concentrations ofchemical compounds required to achieve an activity (C_(ther)), whereinthe activity correlates with a desired therapeutic effect in vivo;predicts cytotoxicity of the compounds according to CATS assayprocedures described herein; determines the ratio of C_(tox):C_(ther)for each compound to provide an Estimated Therapeutic Index (ETI) foreach compound; and prioritizes the compounds as candidate therapeuticagents from the ETIs, wherein a higher ETI correlates with a higherpriority for further development. The use of an estimated TC₅₀ from theCATS assays also would be suitable for generating ETIs and prioritizingcompounds, especially where one is working with a family of structurallyrelated compounds, and the TC₅₀ is from the same particular assay in theCATS battery of assays. (A primary piece of data often used to comparerelative toxicity of compounds is the concentration of drug thatproduces a half maximal effect in any given assay. This value isreferred to as the toxic concentration that produces a 50% response orTC₅₀).

II. Use of Toxicity Cluster Assays to Identify Potential Non-Toxic NewTherapeutics

In certain embodiments, the assays of the presently disclosed andclaimed inventive concept(s) may be used as part of a drug discoveryprogram to identify a putative therapeutic compound with limitedtoxicity. Drug discovery begins with the identification of a range ofcandidate substances that show promise in a targeted therapeutic area.This first step can result in several hundred “hits”. The discovery teamis then faced with the question of which compounds to run in subsequentscreens. CATS analysis at this stage allows teams to prioritize thecompounds based on estimated toxicity or estimated relative toxicityvalues. The top compounds are put through a range of additional screensfor efficacy and specificity. The idea is to identify the core structureor template that shows the most promise for future drug developmentefforts. Once the template is selected, additional chemistry andstructure activity analyses are performed to increase the potency of thecompound. This process yields the lead compounds. A CATS screen at thisstage of the process may be performed to provide toxicity data on thesepotential lead compounds. The top lead compounds are selected to enterpreclinical animal testing. At the animal testing stage, 30% of all drugcandidates fail due to unanticipated toxicity. Incorporation of CATSscreening early in the discovery process should greatly reduce thenumber of compounds that fail during this late stage.

The CATS technique described in the presently disclosed and claimedinventive concept(s) may be employed at any stage in the drug discoveryprogram but is especially valuable early in the discovery process. Theinformation obtained from the cluster analysis provides the chemistswith the appropriate information to design out toxicity, whilemaximizing potency and efficacy in the new templates. In addition, dataobtained from the toxicity cluster analysis can identify subcellulartargets of the compounds that generate the toxicity. Using thesemethods, the putative therapeutic compounds can be ranked or prioritizedbased on their relative toxicities and relative toxicity compared toknown drugs in the same therapeutic and chemical class. For example, theantifungal ketoconazole could be used as a reference compound for newantifungals of the azole class.

High throughput assays for screening numerous compounds for toxicity arespecifically contemplated. In certain embodiments, the high throughputscreens may be automated. In high throughput screening assays, groups ofcompounds are exposed to a biological target. These groups may beassembled from collections of compounds previously individually preparedand since stored in a compound bank, the assembly being random, orguided by the use of similarity programs from which similar structuresare formed.

In addition, there has also been a rapid growth in the deliberatepreparation and use of libraries and/or arrays of compounds. Eachlibrary contains a large number of compounds which are screened againsta biological target such as an enzyme or a receptor. When a biologicalhit is found, the compound responsible for the hit is identified. Such acompound, or lead, generally exhibits relatively weak activity in thescreen but forms the basis for the conduct of a more traditionalmedicinal chemistry program to enhance activity. The libraries may beprepared using the rapidly developing techniques of combinatorialchemistry or by parallel synthesis (DeWitt et al, Proc Natl Acad Sci,90, 6909, 1993; Jung et al, Angew Chem Int Ed Engl, 31:367-83, 1992;Pavia et al., Bioorg Med Chem Lett, 3:387-96, 1993).

Alternatively, the compounds to be screened may be from a library basedupon a common template or core structure [see for instance Eliman andBunin, J Amer Chem Soc, 114:10997, 1992 (benzodiazepine template), WO95/32184 (oxazolone and aminidine template), WO 95/30642(dihydrobenzopyran template) and WO 95/35278 (pyrrolidine template)].The template will have a number of functional sites, for instance three,each of which can be reacted, in a step-wise fashion, with a number ofdifferent reagents, for instance five, to introduce 5×5×5 differentcombinations of substituents, giving a library containing 125components. The library will normally contain all or substantially allpossible permutations of the substituents. The template may be a‘biased’ template, for instance incorporating a known pharmacophore suchas a benzodiazepine ring or an ‘unbiased’ template, the choice of whichis influenced more by chemical than biological considerations.

Thus, the presently disclosed and claimed inventive concept(s) may beused to identify lead compounds for drug discovery. In addition to thelibrary screening discussed above, such lead compounds may be generatedby random cross screening of single synthetic compounds madeindividually in the laboratory or by screening extracts obtained fromnatural product sources such as microbial metabolites, marine spongesand plants.

In another alternative, the compounds may be generated through rationaldrug design based on the structure of known biologically activecompounds and/or their sites of biological action. This has now beencomplemented by the powerful techniques of computer-assisted drugdesign. The goal of rational drug design is to produce structuralanalogs of biologically active molecules of interest. Such technologieswill yield potentially thousands of compounds for a particularindication that may be screened for cytotoxicity using the presentlydisclosed and claimed inventive concept(s).

III. Kits

In certain aspects of the presently disclosed and claimed inventiveconcept(s), all the necessary components for conducting the CATS assaysmay be packaged into a kit. Specifically, the presently disclosed andclaimed inventive concept(s) provides a kit for use in a cytotoxicityassay, the kit comprising a packaged set of reagents for conducting twoor more cell health assays selected from the group consisting of a cycleevaluation assay, mitochondrial function assay, energy balance assay,cell death assay, oxidative stress assay, metabolic activation assay,and metabolic stability assay; wherein said two or more cytotoxicityassays are distinct from each other. In addition to the reagents, thekit may also include instructions packaged with the reagents forperforming one or more variations of the CATS assay of the presentlydisclosed and claimed inventive concept(s) using the reagents. Theinstructions may be fixed in any tangible medium, such as printed paper,or a computer-readable magnetic or optical medium, or instructions toreference a remote computer data source such as a worldwide web pageaccessible via the internet.

While the above embodiments contemplate kits in which there is one assayperformed from each of the classes of cycle evaluation, mitochondrialfunction, energy balance and cell death assays it is contemplated thatthe kits and the methods may involve conducting more than one of anytype of the assay. As such in addition to the kits comprising thereagents for a first, second, third, fourth and fifth assay, it iscontemplated that the kits also may comprise the reagents for conductinga second assay from each of the classes. Therefore, it is contemplatedthat the kits also may comprise the reagents for conducting a pluralityof distinct cell cycle evaluation assays; the reagents for conducting aplurality of distinct mitochondrial function assays; the reagents forconducting a plurality of distinct energy balance assays and thereagents for conducting a plurality of distinct cell death assays.

The presently disclosed and claimed inventive concept(s) alsocontemplates kits constructed for use in any of the specific toxicityscreening assays described herein, including but not limited to,organ-specific screens such as cardiac specific screen, anti-tumorscreen, drug-drug interaction screen, species-specific screen,multi-cell screen, and the like. Such kits would be constructed asdescribed herein above but would contain the reagents necessary for theassays specific to such screens, as described in detail herein. Forexample, but not by way of limitation, a kit for a cardiac specificscreen will include a packaged set of reagents for conducting the two ormore cell health assays as described in detail herein above, as well asa packaged set of reagents for conducting at least one cardiac-specificcell health assay selected from the group consisting of assays ofcardiac hypertrophy, QT interval prolongation, and cardiac cellphysiology. A kit for an anti-tumor screen in accordance with thepresently disclosed and claimed inventive concept(s) may include thepackaged set of reagents for conducting the two or more cell healthassays as described in detail herein above, as well as a packaged set ofreagents for conducting an assay of the expression level of one or moretarget molecules. A kit for a species-selector screen in accordance withthe presently disclosed and claimed inventive concept(s) may include apackaged set of reagents for conducting one or more assays related tometabolic activation, metabolic stability and/or metabolic profiling,and may further include the packaged set of reagents for conducting thetwo or more cell health assays as described herein above. A kit for adrug-drug interaction screen in accordance with the presently disclosedand claimed inventive concept(s) may include a packaged set of reagentsfor conducting one or more assays related to cytochrome P450 enzymeinduction, cytochrome P450 inhibition and/or metabolic activation, andmay further include the packaged set of reagents for conducting the twoor more cell health assays as described herein above.

Introduction to In Vitro Toxicity Screening

There are several key cellular events that can be used to assesscompound toxicity. These include, but are not limited to, loss ofmembrane integrity, mitogenesis, and altered mitochondrial function.Most drug evaluations focus on a single endpoint such as cell viability(live versus dead cells). This approach can lead to false negative orfalse positive results. The screening approach described in thepresently disclosed and claimed inventive concept(s) combines theresults of several biochemical assays to obtain toxicity profiles foreach test compound. Test compounds were evaluated for cytotoxicity inTier 1 (general cell health) screening assays. Each assay was chosenbecause it monitors an important cellular process that can provideinformation on toxicity and on the potential mechanisms of toxicity.

Leakage of intracellular proteins, such as lactate dehydrogenase (LDH),into the outer milieu can provide information on cell death throughdisruption of the cell membrane. The release of α-glutathioneS-transferase (α-GST) was used to monitor membrane integrity or celldeath in this study. In blood, α-GST is a specific liver protein thatprovides information on cell death that is more reliable than therelease of lactate dehydrogenase (LDH) (Vickers, 1994; Redi, 1995). Theprimary reasons for this are as follows: LDH is present in red bloodcells and therefore medium that contains more than 10% serum will havehigh levels of background LDH activity that could mask small changes inrelease from the test cells. The assay is based on enzyme activity,which means that compounds that directly inhibit LDH activity wouldresult in false negative data. In comparison, α-GST is found only inhepatocytes and kidney proximal tubule cells, not in serum. Thus,background levels are extremely low. The α-GST assay is an ELISA thatmeasures protein mass, not activity, and therefore is less likely to beinfluenced by the test compounds. The presence of α-GST in blood samplescollected during in vivo studies indicates toxicity specific to theliver.

Information on cell number relative to controls is important in order todetermine whether or not a compound is acutely toxic or simply slowing,or inhibiting cell replication. Cell number was determined in vitro,using a modified propidium iodide assay or comparable assay such as theCyQUANT® GR-fluorescence assay (Wilson et. al., 1999).

Compounds that alter mitochondrial function or cellular energy balancewill ultimately produce cell death. Therefore it is important to monitorthe effects of the test compounds on mitochondrial function and energybalance. The reduction of tetrazolium dyes such as3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)(Mosmann, 1983; Huveneers-Oorsprong, et al., 1997) and Alamar blue (AB)(Goegan et al., 1995) to chromaphores detectable by spectrophotometricor fluorometric analysis have been used extensively as indicators ofcell viability. Early reports suggested that reduction of these dyesoccurred solely in the respiratory chain of mitochondria (Slater et al.,1963). More recent studies provide strong evidence that othermitochondrial and cytosolic pathways utilizing NADH or NADPH as electrondonors are also involved (Berridge and Tan, 1993; Andrews et al., 1997).Thus, while MTT and AB remain excellent general indicators of cellhealth, they may not be predictive of mitochondrial function in theabsence of supporting data such as ATP or mitochondrial membranepermeability.

When MTT data are combined with information about cell number andmembrane integrity it is possible to develop a more completetoxicological profile. Evaluating a compound in a “panel” of assays withdiverse biochemical endpoints builds redundancy into the screeningprocess and reduces false positive or negative results. In addition,multi-endpoint analysis provides information on relative toxicitybetween test or reference compounds as well as insight into potentialmechanisms of toxicity. This information should provide earlyindications of potential adverse effects of new chemicals early in thediscovery process. This information can then be used to design newcompounds with improved therapeutic indices.

In order to provide the most meaningful and up-to-date toxicity data,current assays may be deleted and or new assays added to the screeningmethods of the presently disclosed and claimed inventive concept(s). Themost complete toxicity profile will be obtained by evaluating testcompounds in at least 4-5 different assays; however, it is possible thatsome evaluations will be done with fewer endpoints. In these instancesevaluation of data should be done with caution and interpretationsshould not go beyond making relative comparisons to other compounds.

Cardiac Toxicity Screening

Adverse events in the heart are of three general types: (1) QTprolongation of ion channel effects, (2) hypertrophy, and (3)cytotoxicity. The processes involved in the mechanical contraction andrelaxation of the heart muscle are complex and are controlled by ionmovement. The electrophysiology of the beating heart includes atrialdepolarization (P-wave), ventricular depolarization (QRS), andventricular repolarization (T). Several drugs have been shown toincrease the QT interval through interference with potassium (K⁺)movement. This can lead to Torsade de Pointe, ventricular fibrillationand sudden death. Because of the serious nature of these events,regulatory authorities have issued requirements for evaluation of newchemical entities for potential QT prolongation prior to regulatorysubmission.

Therefore, the presently disclosed and claimed inventive concept(s)provides a method of determining a level of cardiac specificcytotoxicity by monitoring compound effects on mitochondrial function,cell membrane integrity, oxidative stress, cell mortality, oxidativestress, heart cell viability, heart cell morphology, and cardiac cellphysiology/beat rate.

In such a method of determining a level of cardiac toxicity of achemical compound, cardiomyocytes are typically isolated and establishedas primary cultures. Test compounds are added over several exposureconcentrations, and the following analyses may be performed:mitochondrial function (such as but not limited to, an ATP assay), cellmembrane integrity (such as but not limited to, a GST leakage assay orTroponin I release assay), cell mortality, and oxidative stress assaysand other cell health function assays as described herein. Such assaysare performed as described herein previously. In addition, furtheranalyses performed in accordance with this particular method of thepresently disclosed and claimed inventive concept(s) include thefollowing: cardiac hypertrophy assays, QT interval prolongation assays,and cardiac cell physiology assays, as described in further detailherein below. Once such assays are performed, a level of cardiactoxicity for the chemical compound is determined as described in U.S.Pat. No. 6,998,249 (previously incorporated herein by reference), and/oras described herein previously.

Cardiac hypertrophy refers to an increase in the size of the heart or ina select area of the tissue. Hypertrophy occurs due to an increase inthe size of cells, while the number of cells stays the same. In general,cardiac hypertrophy allows the heart to maintain or increase cardiacoutput as a compensatory response to stress. However, a prolonged stateof hypertrophy can lead to a reduction in ejection-fraction and heartfailure. It is therefore important to evaluate new chemical entities forpotential cardiac toxicity.

In response to hypertrophy there is seen an increased expression ofnumerous cardiac genes, and thus methods of measuring cardiachypertrophy involve detecting the levels of such cardiac hypertrophymarkers. Such methods may measure the levels of mRNA expression orprotein expression by well known methods in the art. Examples of cardiachypertrophy markers include, but are not limited to, atrial natriureticpeptide (ANP), brain natriuretic peptide (BNP), skeletal α-actin, C-fosand C-jun, and the like.

The term “QT interval” as used herein will be understood to refer to ameasure of the time between the start of the Q wave and the end of the Twave in the heart's electrical cycle. The QT interval is thus dependenton the heart rate (the faster the heart rate, the shorter the QTinterval). If abnormally prolonged or shortened, there is a risk ofdeveloping ventricular arrhythmias.

QT interval prolongation may be measured utilizing assays that measurethe disruption of ion channels. One example of such type of assay is ahERG channel assay.

The HERG channel assay is described herein as associated with anindication of QT interval prolongation, and such assay is also a primaryindicator of K⁺-channel blockage. HERG (which stands for “HumanEther-a-go-go Related Gene”) encodes a potassium ion channel responsiblefor the repolarizing I_(Kr) current in the cardiac action potential.This channel is sensitive to drug binding, which can result in decreasedchannel function and the so-called acquired long QT syndrome. Althoughthere exist other potential targets for adverse cardiac effects, thevast majority of drugs associated with acquired QT prolongation areknown to interact with the HERG potassium channel. One of the mainreasons for this phenomenon is the larger inner vestibule of the HERGchannel, thus providing more space for many different drug classes tobind and block this potassium channel. Therefore, as mentioned above,regulatory authorities have issued requirements for evaluation of newchemical entities for potential QT prolongation prior to regulatorysubmission.

It is to be understood that the presently disclosed and claimedinventive concept(s) is not limited to the particular HERG inhibitionassay described herein. Other methods of measuring QT-prolongation knownin the art also fall within the scope of the presently disclosed andclaimed inventive concept(s).

Methods of measuring cardiac cell physiology may be monitored by anymethods described herein or known in the art. In particular, cardiaccell physiology may be measured by determining the percentage of beatingcells or the beat rate per 30 second intervals.

In a further embodiment, a method of determining cardiac specifictoxicity is provided. In such method, freshly isolated liver cells arealso provided and subjected to the same assays and analyses as thecardiomyoctes. This will allow for an identification of target organspecificity for chemical compounds that are specifically toxic tocardiac cells. In the method, the analyses from the cardiomyocytes andthe liver cells are compared, and it is determined that the chemicalcompound is more toxic to cardiac cells than non-cardiac cells if theC_(tox) for the cardiac cells is less than the C_(tox) for thenon-cardiac cells.

FIG. 1 illustrates an evaluation of the drug adriamycin in the dual cellCardiotox model of the presently disclosed and claimed inventiveconcept(s). By comparing concentration response analyses of membranepermeability (MemTox), MTT, GSH and ATP assays, it can be seen thatadriamycin is much more toxic to heart than to liver (see arrows). It isclear that cell viability as determined by membrane integrity issignificantly lower in heart cells than at the same exposureconcentration in liver cells. Markers of early toxicity such as MTT andGSH are also more sensitive in heart cells than in liver. Exposureconcentrations, serum protein, and time are constant in both cell types.These date indicate that the heart would be more sensitive to adriamycintoxicity than liver.

In FIG. 2, concentration response analyses for Imatinib (GLEEVEC®) inliver cells are shown. Imatinib is a tyrosine kinase inhibitor used inthe treatment of chronic myelogenous leukemia, and such drug haspreviously been associated with cardiac toxicity, which was hypothesizedto occur by mitochondrial damage. Analyses of Imatinib in the H4IIEliver cell line reveals that mitochondria is indeed the most sensitivetarget. In heart cells, the drug is three to four-fold more potent. Bycomparing multiple drugs that target tyrosine kinase, a comparative dataset can be used to add even more predictive information.

FIGS. 3-5 are directed to a CardioTox screen of the drugs CELEBREX®(FIGS. 3, 3A, and 5) and Vioxx (FIGS. 4, 4A, and 5A). Both drugs inhibitCOX-2, and Vioxx was voluntarily removed from the market following thediscovery of an increased incidence of cardiac toxicity. Both CELEBREX®and Vioxx would be considered safe based on the in vitro toxicityscreen, which utilized measurements of membrane permeability (MemTox),MTT, and ATP. However, Vioxx increased fat accumulation and oxidativestress and was more effective at inducing the hypertrophy marker ANP(FIGS. 5 and 5A). Differences in the toxicity profiles suggest thatthese effects are not related to the target.

The CardioTox screening panel is designed to provide information on theeffects of new drug candidates on QT prolongation and can provideinformation on the test compound's propensity to produce cardiactoxicity relative to liver toxicity. The cluster of endpoints evaluatedexemplifies the systems biology approach to in vitro screening and isthe first model that accurately assigns risk for organ specific toxicitydifferentiating heart from liver adverse effects.

While the above-referenced screen has been described with reference todetermining a level of cardiac toxicity, it is to be understood that thepresently disclosed and claimed inventive concept(s) is not limited tomethods of determining cardiac toxicity, but rather that the methods ofthe presently disclosed and claimed inventive concept(s) may be utilizedto determine organ-specific toxicity for any desired organ. Based on themethods described herein above and below with reference to screening ofmultiple cell types, one of ordinary skill in the art will easily beable to adapt the methods described herein with reference to cardiactoxicity for use with determining levels of organ-specific toxicity inother tissues and organs, and therefore such methods also fall withinthe scope of the presently disclosed and claimed inventive concept(s).

Anti-Tumor Screen

The presently disclosed and claimed inventive concept(s) providesmethods for determining a level of toxicity for an anti-tumor drug. Mostanti-tumor drugs are designed to either be cytostatic (i.e., suppresscell growth and multiplication) or cytotoxic (cause cell death). If adrug is cytostatic, it is important to understand the “off-targettoxicity” of the anti-tumor drug. If a drug is designed to be cytotoxic,then the evaluation of off-target toxicity becomes even more difficult.In both cases, it is important to be able to differentiate toxicity dueto intended pharmacology and toxicity related to chemistry.

The primary mechanism of action of a cytostatic anti-tumor drug focuseson targets that are specific to tumor cells. In contrast, cytotoxicanti-tumor drugs target general processes, by directly interacting withDNA or disrupting cell division processes. Tumor cells are moresensitive than normal cells due to their higher rate of division, butfor these drugs a considerable amount of non-specific toxicity occurs.Some of the many side effects of cytotoxic drugs include, but are notlimited to, hematoxicity, nephrotoxicity, hepatotoxicity, andneurotoxicity, and such side effects are common.

In the Anti-tumor screen of the presently disclosed and claimedinventive concept(s), at least three different cell types are utilized:(1) tumor derived cells, (2) proliferating cells from normal tissue, and(3) non-proliferating cells from normal tissue. The evaluation ofanti-tumor agents in multiple cell types provides information onmechanism-based toxicity versus efficacy. In addition, it may bedesirable to include more than three cell types. For example but not byway of limitation, it may be desirable to include more than one type oftumor cell, to determine a drug's efficacy/potency against multipletypes of tumors. In addition, it may be desirable to include a specificcell type from more than one species (i.e., hepatocytes from rat andhuman) to identify species differences in potency/efficacy and/ortoxicity of the anti-tumor agent.

The anti-tumor agent can be evaluated in a toxicity screen of keybiochemical functions (as described in detail herein above), withemphasis placed on cell proliferation assays as well as biochemicalfunction and cell viability. Comparisons can then be made toquantitative markers in the concentration response curves constructedwith the toxicity data. For example, comparisons of TC₅₀, TC₂₀ and TC₉₀values can prove very useful in the anti-tumor screen of the presentlydisclosed and claimed inventive concept(s).

The methods of the presently disclosed and claimed inventive concept(s)provide the ability to separate target from off-target toxicity, whichis essential when evaluating an anti-tumor agent. This is performed bydetermining the relative abundance of the intended target in each of theat least three cell types. The toxicity response calculated in theprevious paragraph can then be normalized to the target in each cell.

The advantages of the Anti-tumor screen of the presently disclosed andclaimed inventive concept(s) are four-fold: (1) the use of at leastthree cell types (tumor cell, normal proliferating cell and normalnon-proliferating cell); (2) the ability to identify target versusoff-target effects; (3) the ability to examine the potency/efficacy andtoxicity of the anti-tumor agent in multiple species; and (4) theability to examine the potency/efficacy and toxicity of the anti-tumoragent in multiple types of tumors.

In the Anti-Tumor screen illustrated in FIG. 6, cell number and GSTassays are shown that demonstrate that the anti-tumor drug is highlytumor cell specific. These results clearly demonstrate that theanti-tumor drug is specifically toxic to the H4IIE tumor cell line, butis not toxic to the rat primary hepatocytes or the normal rat kidney(NRK) cells.

FIG. 7 illustrates the advantages of using multiple cell types in theanti-tumor screen of the presently disclosed and claimed inventiveconcept(s). This figure illustrates the ability to examine multipletumor cell types in a single screen (i.e., SK-MEL28, C32). Note that thetwo tumor cell types were affected more than the non-tumor cells. Thisfigure also illustrates that ability to identify species-specificdifferences in toxicity. For example, rat hepatocytes were verysensitive to the drug, whereas the drug was much less toxic to humanhepatocytes. This demonstrates that a rat would not be the best in vivomodel to evaluate the safety of this drug, as the results would overestimate toxicity.

FIGS. 8 and 8A-8H illustrate an anti-tumor screen of the drug Cisplatinin H4IIE tumor cells (FIGS. 8, 8A, and 8B), rat primary hepatocytes(FIGS. 8F-8H), and NRK (normal rat kidney) cells (FIGS. 8C-8E).Cisplatin is a DNA alkylating agent that exhibits renal toxicity. It ishypothesized to act through mitochondrial toxicity as well as apoptosis.The analyses shown in FIGS. 8 and 8A-8H illustrate that the liver tumorcell line (H4IIE) is the most sensitive model. All markers exceptviability (open squares) are reduced, and apoptosis (bottom graph)showed the greatest increase relative to the other cell systems. The NRKcells were most sensitive to cell proliferation, with a moderateincrease in apoptosis. The primary hepatocytes model showedmitochondrial effects at higher exposure concentrations with minorchanges in apoptosis. Therefore, the anti-tumor screen of the presentlydisclosed and claimed inventive concept(s) confirms the anti-tumoractivity of Cisplatin, and shows that a cytotoxic drug has its greatestpotency against tumor cells and cells undergoing replication.

FIGS. 9 and 9A-9H illustrate an anti-tumor screen of the drugMethotrexate in H4IIE (FIGS. 9, 9A, and 9B), rat primary hepatocytes(FIGS. 9F-9H), and NRK cells (FIGS. 9C-9E). Methotrexate inhibits folatesynthesis, and its toxicity is hypothesized to occur throughmitochondrial damage. The analyses shown in FIGS. 9 and 9A-9H illustratethat the tumor cell line is most sensitive to mitochondrial damage, asdetermined by the concentration dependent changes in ATP, MTT, and cellnumber. The mitochondrial damage caused a release of cytochrome c whichin turn activated apoptotic pathways. The normal dividing cells (NRK)and the normal non-dividing cells (primary hepatocytes) wereconsiderably less sensitive to these effects.

FIGS. 10 and 10A-10H illustrate another anti-tumor screen of thepresently disclosed and claimed inventive concept(s), utilizing the drugDoxorubicin. Also known as Adriamycin, Doxorubicin is a topoisomeraseinhibitor that exhibits a general toxicity, including cardiac toxicity(see FIG. 1 above), myelotoxicity, and hepatic and renal toxicity.Doxorubicin's toxicity is hypothesized to occur through mitochondrialdamage and oxidative stress. FIGS. 10 and 10A-10H illustrate that thiscytostatic drug is toxic to all of the cells tested, and this isconsistent with its reported toxicity. When the data from the anti-tumormodel are combined with data obtained from the cardiotox model describedabove, it is clear that this cytotoxic drug is toxic to many tissuetypes, with the greatest degree of potency observed in heart cells.

FIGS. 11 and 11A illustrate the ability of the presently disclosed andclaimed inventive concept(s) to perform “target profiling” anddifferentiate target versus off-target effects and cell sensitivity ofanti-tumor agents. Different cell lines and primary cells can haveconsiderably different levels of the intended target of the anti-tumordrug. If toxicity assays are performed in multiple cell types withoutunderstanding the relative abundance of the target protein, cells with ahigher level of target may appear more sensitive to toxicity when infact the drug is acting as it was designed to do. Thus, to understandtoxicity not related to the intended target, the toxicity of the testcompound as measured by the TC₁₀, TC₅₀, or TC₉₀ is normalized to theamount of target present. FIG. 11 shows that the amount of target in theH4IIE and primary hepatocytes is similar. In contrast, the NRK cellspossess significantly more of the target mRNA. By normalizing the TC₅₀values for viability to the expression of target, it is clear that theH4IIE cells are less sensitive to toxicity than the primary hepatocytes(FIG. 11A). This is not to say that the H4IIE cells are not responsive(note they are at 50% viability); they simply are more resistant to celldeath than the primary hepatocytes. The difference in acute toxicitybetween the H4IIE and primary hepatocytes can be attributed tooff-target toxicity. The NRK cells were most sensitive to the drug, butthe toxicity observed could not be differentiated from the intendedeffect of the drug in the presence of its target. The additionalinformation provided by the target profiling experiment indicates thatthe target is ubiquitous in nature, and as such drugs designed tointeract with this target would be expected to have toxicity in multiplecell types.

Species-Specific Screen

An important component of any new drug evaluation is the potential forspecies specific toxicity. Rodent studies may show no adverse signs,while a non-rodent species may have severe or even lethal toxicity. Whenthis situation occurs, investigators must begin to ask questionsregarding the underlying mechanism, the differences between species, andwhich species is most relevant to human exposure and potential toxicity.On occasion, the animal tests must be repeated. Answering thesequestions is costly and time consuming for the company and can delay thedelivery of a potentially good drug to market. The ability to screen newdrug candidates for potential species-specific toxicity can helpscientists select the best and most appropriate second species foranimal safety evaluations. More importantly, this information can formthe basis of mechanism-based species differences which in turn canprovide the most relevant data for human safety assessment communicationto the FDA.

The Species-Specific screen of the presently disclosed and claimedinventive concept(s) focuses on metabolic profiles and how they mayaffect toxicity profiles. The process involves a sequential evaluationof various processes and can be done with any combination of desiredanimal systems, such as but not limited to, rat, dog, monkey, rabbit andhuman systems.

In the first step, the metabolic stability of a chemical compound isdetermined in each species. This analysis determines the rate ofmetabolism in hepatic microsomes from the species being evaluated.

FIG. 12 illustrates a metabolic stability assay performed in accordancewith the presently disclosed and claimed inventive concept(s). Themetabolism of Propanolol is examined in liver microsomes of rat andhuman. It is clear that the compound is metabolized to a much greaterdegree in rat than in human microsomes. The graph on the left depictsrates of metabolism by examining loss of parent over time, while thegraph on the right provides basic information on metabolic stabilityexpressed as the amount of parent remaining at the end of the reactionperiod. The reactions include a single co-factor (NADPH) for phase Imetabolism and a second co-factor for glucuronidation or Phase IImetabolism if it is required.

In the second step, the metabolic activation or the formation ofreactive intermediates is determined in each species. This analysisdetermines the species specific formation of electrophiles. The systemutilizes hepatic microsomes from the species being evaluated. In a novelassay of metabolic activation that is also encompassed within the scopeof the presently disclosed and claimed inventive concept(s), a knownamount of reduced glutathione (GSH) is added with the chemical compound,and the disappearance of free GSH is monitored using a labeled probe.The GSH pool decreases in direct proportion to a cytochromeP450-mediated increase in electrophilic intermediates. In a second stepof the assay, an inhibitor of cytochrome P450 (such as but not limitedto, ABT) is added to the assay, and the decrease in the GSH pool is notobserved, thereby confirming that the decrease is due to the cytochromeP450-mediated increase of electrophilic intermediates.

FIG. 13 illustrates the evaluation of drugs with structural alerts formetabolic activation. Acetaminophen (APAP) is included as a control drugthat is known to produce reactive intermediates upon metabolism (bars onfar left). Note that in the presence of ABT, the loss of GSH is notseen. The drugs on the far right of the graph are all known to undergometabolic activation. Liver microsomes from Phenobarbital/BNF inducedrats were used in the upper graph. As expected, all of the compoundsproduced a reduction in GSH, but tacrine and flutamide had the mostpronounced effects. The formation of reactive intermediates was greaterin the induced rat microsomes than in normal human microsomes (bottomgraph). These data illustrate the importance of evaluating speciesspecific metabolism and also the ability of the assay to identify drugsthat undergo metabolic activation.

In the third step, a qualitative evaluation of the metabolic profilesproduced in microsomes from each species is performed. Profiles fromeach species are compared, and peaks present in one but not the other isflagged.

FIGS. 14 and 15 illustrate the analyses of metabolic profiles for ratand human hepatic microsomes in the presence of propanolol by LC/MS inion selective mode. When human and rat Tsixty profiles are compared inFIG. 14, it is evident that a peak exists between 2.78 and 3.25 in ratthat is not present in human. Likewise, in FIG. 15, there are peakspresent in the rat Tsixty TIC, M+16 and M+32 that are not present in thehuman Tsixty TIC, M+16 and M+32. These data indicate the presence of ametabolite in rat that is not present in human.

In the fourth step, the test compound is evaluated in primaryhepatocytes from each species against a panel of two or more biochemicalmarkers for cell health. This toxicity is performed as described indetail herein above. Shifts in the toxicity profiles can then be relatedto changes in metabolism. Taken together and in series, these assaysprovide a comprehensive picture of potential species specific toxicitylinked to metabolism.

IV. Examples

Examples are provided hereinbelow. However, the presently disclosed andclaimed inventive concept(s) is to be understood to not be limited inits application to the specific experimentation, results and laboratoryprocedures. Rather, the Examples are simply provided as one of variousembodiments and is meant to be exemplary, not exhaustive.

Example 1 In Vitro Toxicity Screening of Compound A in RatCardiomyocytes and Rat Hepatoma (H4IIE) Cells: 1, 3, 6, and 24 HourExposures

One test compound (Compound A) was received as dry powder and evaluatedfor potential cytotoxicity in a battery of in vitro assays. The compoundwas tested using both rat cardiomyocytes (CM) from rat neonates and arat hepatoma (H4IIE) cell line. The cells were seeded into 96-wellplates and cultured in medium containing 20% bovine serum. Following anequilibration period of 48 hr, the cells were treated with the testcompound at concentrations of 0, 1, 5, 10, 20, 50, 100, and 300 μM for amaximum of 24 hr (overnight) at 37° C. in 5% CO₂. Camptothecin androtenone were included as positive control compounds. The cellsupernatant or the cells themselves were harvested for biochemicalanalysis. General cytotoxicity was evaluated by monitoring membraneintegrity, mitochondrial function, cell proliferation, oxidative stress,and apoptosis. In addition, interaction with P-glycoprotein (PgP) onH4IIE cells and solubility of the test compound were also assessed. Themeans of each exposure group (n=3-7) were calculated for each assayperformed. Intra assay variation well-to-well was typically less than10% with plate to plate variation typically less than 20%.

The responses obtained in the assays were not normalized to cell numberor membrane leakage (MemTox). Thus, for correct interpretation ofresults, all data must be visually normalized within the graphs in orderto ascertain whether the change was directly due to the test compound orthe result of difference in cell numbers. Cell number and cell viabilityare key parameters for correct interpretation of results.

The results are summarized in Tables 1-4 and in FIGS. 16-61. Table 1compares the test compound TC₅₀ values for each assay endpoint. Testcompounds are rank-ordered from most to least toxic based on their TC₅₀values and overall shape of the response curves. The C_(tox) value(H4IIE), or estimated sustained blood concentration where toxicity wouldfirst be expected to occur in a rat 14-day repeated dose study, is alsoincluded in this table. Negative and positive controls are included withevery run. Assay response is continually monitored to assure reliableresults. Camptothecin and rotenone were included as positive controlsfor all endpoints (FIGS. 44 and 60 for rotenone and FIGS. 45 and 61 forcamptothecin), while DMSO at 0.5% in culture medium was included as anegative control. Table 2 summarizes oxidative stress and apoptosisdata, Table 3 summarizes the solubility data, and Table 4 providesinformation on interaction with PgP.

The C_(tox) (H4IIE) value was developed using 24 hr in vitro toxicitydata and was validated by retrospectively evaluating compounds acrossdifferent classes of drugs in 14-day rat studies in which compounds wereadministered on a daily basis and pharmacokinetic data was available.These evaluations showed that the C_(tox) value was an accurateprediction of in vivo toxicity. Thus, C_(tox) values are only determinedfor the 24 hr in vitro results. The 6 hr toxicity analyses are typicallynot performed but are beneficial for interpretation of results whencompound toxicity is high (e.g., Ctox≦20 μM).

The present example was designed to evaluate the relative toxicity of aHep-C protease inhibitor identified as Compound A compared to twoclasses (anthracyclines and antiretroviral) of drugs currently on themarket that have been associated with cardiac or liver toxicity.

Freshly isolated primary culture of rat neonate cardiomyocytes and a rathepatoma cell line were used. The endpoints measured in this study werechosen because they represent pivotal points in pathways controllingcell health. The two cell models were selected to provide an estimate ofpotential cardiac versus systemic toxicity. By comparing the effectsobserved with Compound A to those observed for several anti-tumor andanti-viral drugs an added perspective regarding the predicted in vivotoxicity of the test compound can be achieved.

Adriamycin was selected as a reference compound because it is a wellknown anthracycline member causing cardiac toxicity (Horenstein et al,2000). The cardiac toxicity observed following anthracycline treatmenthas been linked to the production of reactive oxygen species resultingin depletion of reduced glutathione (GSH), and an increase in membranelipid peroxidation resulting in damage to cellular macromoleculesincluding mitochondria. The heart appears to be more sensitive to theseeffects because it has relatively poor antioxidant defense mechanismsrelative to other organs such as liver.

The second group of drugs used as reference compounds comprise a widerange of anti-viral drugs including protease inhibitors (PI) such asritonavir, lopinavir, and indinavir (Esposito et al, 2006; Oldfield andPlosker, 2006; Von Hentig et al, 2006) (FIGS. 36 and 53; 38 and 55; and41 and 57, respectively) non-nucleoside reverse transcriptase inhibitors(NNRTIs) which include efavirenz, delavirdine, and nevirapine(Perez-Elias et al, 2005) (FIGS. 37 and 52, 39 and 54 and 42 and 58,respectively), and nucleoside reverse transcriptase inhibitors (NRTIs)which include abacavir and AZT (Sriram et al, 2006) (FIGS. 40 and 56 and43 and 59, respectively). Drugs that resemble nucleosides cause areduction in mitochondria density which leads to cytotoxicity. One ofthe mechanisms underlying this effect is inhibition of mitochondrial DNApolymerase gamma which in turn prevents mitochondrial replicationresulting in a reduced number of mitochondria in tissue. Typically thisis a delayed effect that requires at least one doubling time in order tobe detected in vitro (Martin et al, 1994; Lewis and Dalakas, 1995).

The present study had four primary objectives: (1) to evaluate aprotease inhibitor drug for Hep-C treatment in cell based modelsdesigned to predict in vivo toxicity; (2) to determine if the heartcould be a more sensitive target organ of toxicity; (3) compare the testdrug's effects to those obtained for several approved anti-tumor andanti-viral drugs currently on the market; and (4) to identify potentialmechanism(s) of toxicity.

Table 1 summarizes the acute toxicity biochemical effects incardiomyocytes at 1, 3, 6, and 24 hr. Table 2 summarizes the chronictoxicity markers (glutathione (GSH) and membrane lipid peroxidation)over the same time points. Adriamycin in cardiomyocytes producedsignificant changes in mitochondrial function as measured by ATP and MTTat 3 and 6 hr and these effects preceded cell death (Table 1; FIGS. 21Aand 22A). After a 3 hr exposure to adriamycin the most sensitivesubcellular targets in the cardiomyocytes were mitochondria andreduction in reduced glutathione (GSH). More than 90% of the cellularGSH had been depleted after 3 hr (Table 2). Cell death occurred by 6 hrof exposure with nearly all cells dead after 24 hr (Table 1 and Table 2;FIGS. 21, 22, and 23). Compared to adriamycin, the test compoundCompound A was approximately 64-fold less potent in the MTT assay,16-fold less potent in the ATP assay and more than 40-fold lesseffective at inducing cell death (Table 1 summary of rat cardiomyocytedata). This trend was also true when Compound A was compared to theother reference anthracyclines (idarubicin, mitoxantrone, daunorubicin,pirarubicin, and epirubicin) (Table 1 summary of TC₅₀ values in ratcardiomyocytes and FIGS. 31-35 and 46-51). Although the mitochondrialmarkers were most sensitive following exposure to Compound A, the testcompound's potency was much lower compared to the anthracyclines. Inaddition, the test compound (Compound A) did not deplete GSH levelsuntil the highest exposure concentrations were reached (FIGS. 16-18).There was a compensatory increase in GSH levels observed in themid-exposure ranges that was observed at all time points tested. Thisincrease in GSH was most likely due to induction of GSH-synthetaseactivity which has been demonstrated for other drugs and chemicals. Theinduction of GSH-synthetase activity suggests some stress on the celland indicates a protective or adaptive response by the cell. The resultsfurther indicate a mechanism of effect for Compound A different from theanthracyclines. Compound A did not cause cell death under the conditionstested, making it considerably less toxic in the heart thananthracyclines (Table 1). When compared to other PIs, Compound A wasless toxic than ritonavir, and lopinavir and slightly more toxic thanindinavir (Table 1; FIGS. 19, 36, 38 and 41). All of the PIs reduced GSHlevels prior to cell death in the cardiomyocytes and the most toxic PIs,ritonavir caused the most pronounced loss of GSH (FIG. 36).

The ability of the test compound Compound A and adriamycin to induceatrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP)gene expression as markers of cardiac hypertrophy was measured.Hypertrophy of the heart ventricular myocytes is regulated by a complexseries of intracellular signaling processes involving angiotensinreceptors and a series of down stream gene expression changes (Li et al.2006). Endothelin-1 (ET-1) induces hypertrophy of cardiomyocytes in vivoand in vitro. The mechanism of ET-1-induced hypertrophy consists in theactivation of phospholipase C, protein kinase C, extra cellular signalregulated kinase (ERK) and upregulation of cfos and cjun. The expressionof ANP and BNP are upregulated in response to ET-1 (Li et al. 2006).

Expression of ANP and BNP mRNA levels was determined by bDNA analysis ofmRNA. ET-1 at 0.1 μM was included as a positive control. Adriamycin hadno effect on the expression of either peptide (FIGS. 27A and 27B). Thetest compound Compound A increased expression of ANP and BNP in a doseand time dependent manner (FIGS. 25A and 25B). In terms of time ofexposure, 3 and 6 hr were optimal. BNP mRNA was increased 7-fold overcontrols following exposure to 20 μM Compound A (FIG. 25B), while ANPwas increased approximately 2-fold (FIG. 25A). The positive control ET-1at an exposure concentration of 0.1 μM increased ANP expressionapproximately 2-fold after 3 hr (FIG. 25A) and BNP approximately4.5-fold after 3 hr (FIG. 25B).

Nitric oxide was also evaluated since it has been implicated in manypathways related to oxidative stress, apoptosis, and cardiachypertrophy. More specifically, adriamycin induced mitochondrialtoxicity is reduced in the presence of increased iNOS. Thus, acompensatory response to mitochondrial toxicity may be an increase iniNOS (Chaiswing et al., 2005). In this example, rat cardiomyocytesexposed to adriamycin induced iNOS expression more than 15-fold (FIG.27C). There was no detectable increase in iNOS gene expression afterexposure to the test compound Compound A (FIG. 26C) in cardiomyocytes.

In the hepatoma cell model, the test compound showed similar trends inits toxicity profile; however, the liver cell model was considerablyless sensitive to these effects. Serum concentrations were the same inboth cell models so differences in cell type sensitivity cannot beattributed to the presence of different protein concentrations. Theheart model is providing biochemical information unique to heart thatmay provide insight into the risk of cardiac related toxicity in vivo.

Adriamycin was approximately 5-fold less toxic in liver cells than inthe cardiomyocytes. This is based on the relative differences in TC₅₀values for membrane integrity of 33 μM in liver versus 7 μM in myocytes.Effects on mitochondrial markers such as MTT and ATP showed that theformer was affected approximately 13-fold less in liver cells comparedto myocytes (Table 1 H4IIE data), while the later was affected to asimilar magnitude (Table 1 summary of H4IIE data). When cell death wasused as the marker, adriamycin was about 2.5-fold more potent in theheart cells than in liver cells (Table 1; FIGS. 23 and 30). Adriamycinproduced a significant increase in caspase 3 activity in the liver cellline, a committed step in apoptosis. This change was not observed in thecardiomyocytes (FIGS. 19 and 30). The absence of caspase 3 activity inthe cardiomyocytes may have been due in part to the protective effect ofBcl2, which was induced 6-fold after 6 hr at 10 μM and is known toinhibit apoptosis (FIG. 28B). It should be noted that this increase inBcl2 occurred prior to cell death (FIG. 22). Bcl2 is often induced inresponse to BAX a protein that promotes apoptosis. A 3-fold increase inBAX was observed after 1 hr and at an exposure concentration of 10 μM(FIG. 28A). The peak exposure concentration that produced Bcl2expression was lower than the concentration that produced the greatestincrease in caspase 3 activity in the liver cell line.

The production of reactive oxygen species (ROS) can be measured using2′-7′ dichloro-dihydrofluorescein diacetate (DCFDA). Fluorescence signalis directly proportional to production of ROS (Fabiani et al., 2005;Mracek et al., 2006; Gonzalez et al., 2006). The test compound CompoundA produced a small increase (approximately 2-fold) in DCFDA fluorescenceat exposure concentrations of 5 and 10 μM, but this increased to morethan 6-fold after a 24 hr exposure to 50 μM. If these data are comparedto those depicted in FIG. 29 for toxicity in the liver cell model, theincrease in ROS occurs at an exposure concentration just below the firstconcentration to produce impaired mitochondrial function. This resultsuggests that the increase in ROS occurred either just prior toperturbation of mitochondrial function or concomitantly. Adriamycinproduced only small changes in ROS related fluorescence at exposureswhere cytotoxicity was minimal. This is apparent by comparing FIG. 24Bto FIG. 30. The 4-fold increase at 5 μM occurs as mitochondrial damageis beginning.

Several antiviral drugs are substrates for P-glycoprotein transporter(PgP). Interaction of the test compound as well as the referencecompounds with PgP was assessed and the data shown in Table 4 (summaryof PgP binding H4IIE cells). The test compound also had PgP interactionsof approximately the same magnitude as the other antiviral compounds.

In summary, utilization of a two cell model showed distinct differencesin cell sensitivity. The toxicity observed for reference compounds andthe underlying mechanisms was consistent with those reported in theliterature. Thus, the two model approach provided insight into potentialissues related to cardiac toxicity that would not have been identifiedin the liver cell model. The test compound Compound A was less toxic tohepatoma cells than to cardiomyocytes. The mitochondria and reduction inreduced GSH seem to represent the most sensitive targets in both cellmodels. However, for a similar test compound concentration, effects onmitochondrial function markers in the liver cell model were 2-to-3 foldlower than those observed in the heart model, based on a comparison ofTC₅₀ values (Table 1). The Compound A compound induced ANP and BNPexpression indicating some potential to cause cardiac hypertrophy.

Materials and Methods

Experimental Protocol: The test compound was provided and stored at 4°C. until needed. Dosing solutions were prepared in complete culturemedium. The rat hepatoma (H4IIE) cell line was used as the test system.Cells were seeded into 96-well plates and allowed to equilibrate forapproximately 48 hr. Following the equilibration period the cells wereexposed to the test compound at concentrations of 0, 1, 5, 10, 20, 50,100, and 300 μM. Solubility was determined by Nephalometry techniquesimmediately after dosing and prior to harvesting the cells at 6 or 24hr. Following the exposure period, the cells or their supernatant(culture medium) were analyzed for changes in cell proliferation,membrane leakage, mitochondrial function, oxidative stress, andapoptosis. The resultant exposure concentration response curves weregraphed and analyzed for determining the concentration that produced ahalf maximal response or TC₅₀.

Test and Control Articles: The test compounds were received dry or as aliquid and were used to prepare 20 mM stock solutions in DMSO. Thisstock was diluted in DMSO to prepare 0.2 mM stock solutions. Both the 20mM and 0.2 mM stocks were used to prepare dosing solutions of 0, 1, 5,10, 20, 50, 100, and 300 μM in culture medium. The final concentrationof DMSO in the 0-100 μM solutions was 0.5% and at the 300 μM solutionDMSO was 1.5%. The final dosing solutions in medium were prepared on theday prior to dosing. The solutions were wrapped in foil or shielded fromlight and stored at 4° C. until needed.

The details of the preparation and dilutions can be found in thelaboratory. All experiments used dimethylsulfoxide (DMSO) as the testarticle solvent and the negative (solvent) control.

Negative controls of medium plus DMSO (0.5%) were included with andwithout cells. A positive control for complete cell death received 1 mMdigitonin in medium on the day of dosing.

Reagents and Solutions: All chemicals used were reagent grade or better.

Test System: H4IIE Cell Line: Rat cardiomyocytes from rat neonates andrat hepatoma derived H4IIE cells were used as the test systems. Theculture medium used for these cells was Eagles Minimum Essential Mediumwith 10% bovine serum and 10% calf serum. Certified bovine serum andcalf serum were from In vitrogen.

Description of Experimental Setup and Biochemical Assays: Flat bottom96-well plates were seeded with 10,000 cells/well 48 hr prior to dosing.On the morning of the third day after seeding, the test compounds inmedium were added to the plates (DMSO=0.5%). The 300 μM treatment had afinal DMSO concentration of 1.5%.

During method development experiments, it was determined that the 48 hrcell growth period allows cells to move into a stable growth phase priorto treatment. In addition, the effect of DMSO on cell proliferation,MTT, and α-GST was evaluated at DMSO concentrations ranging from 0.05 to4%. These studies showed no effects on any of the endpoints tested atconcentrations below 2%. Finally, the ability of DMSO to enhance celluptake and hence toxicity of a compound was also evaluated. Nosignificant differences in toxicity were detected when a broad range ofketoconazole and amphotericin concentrations were tested at final DMSOconcentrations of 0%, 0.5% and 1.5%.

All of the assays described below may not have been used to evaluate thecompounds submitted. Table 1 provides information on the assays used inthis example.

Cell Proliferation: Cell proliferation in each well was measured withpropidium iodide (PI). This specific nucleic acid binding dye fluoresceswhen intercalated within the nucleic acids. The 15 nm shift enhances PIfluorescence approximately 20 times while the excitation maxima areshifted 30-40 nm. During method development experiments, it wasdetermined that Triton-X-100 was the best solution to permeabilize theH4IIE cells thereby allowing the PI access to intracellular RNA and DNA.Fluorescence was measured using a Packard Fusion plate reader orequivalent reader at 540 nm excitation and 610 nm emission.

Bromodexoyurine (BrDU) Incorporation: This assay was used to monitorcumulative changes in DNA replication. The assay uses the thymidineanalog BrdU to measure S-phase of cell replication and is considered tobe the Gold Standard for cell proliferation. BrDU (10 μM) was includedin the test compound dosing solution to allow BrDU incorporation duringcell replication. BrDU was included for the duration of the exposure, 24hr. After completion of exposure period, the cells were fixed andlabeled with mouse monoclonal anti-BrDU (FITC). Fluorescence wasmeasured using a Packard Fusion or equivalent plate reader at 485 nmexcitation and 530 nm emission.

Membrane Leakage (α-Glutathione S-transferase and/or Adenylate Kinase):The presence of Adenylate Kinase (AK) or α-Glutathione S-transferase(α-GST), both enzyme leakage markers, was measured in the culture mediumusing an activity assay for Adenylate Kinase and/or an ELISA assay forα-GST. At the end of the exposure period, the medium covering the cellsin each well was removed and placed into new 96-well plates withappropriate labeling. These plates containing culture medium were eitheranalyzed immediately or stored at −80° C. until needed for analysis.Luminescence (AK) values were measured with a Packard Fusion orequivalent plate reader, and absorbance (α-GST) values were measuredwith a Packard SpectraCount™ or equivalent reader at 450 nm andreference absorbance at 650 nm.

Tetrazolium Dye Reduction: 3-[4,5-dimethylthiazol-2-yl]2,5diphenyltetrazolium bromide (MTT): After the medium was removed from aplate for α-GST analysis, the cells remaining in each well wereevaluated for their ability to reduce soluble-MTT (yellow) toformazan-MTT (purple). An MTT stock solution was prepared in completemedium just prior to use and warmed to 37° C. in a water bath. Once themedium was removed from all wells, MTT solution was added to each welland the plate was allowed to incubate at 37° C. for 3-4 hr. Internalmethod development experiments have demonstrated that color developmentis linear over this time.

After the 3-4 hr incubation, all medium was removed and the purpleformazan product was extracted using anhydrous isopropanol. Sampleabsorbance was read at 570 nm and reference absorbance at 650 nm with aPackard Fusion or equivalent plate reader.

Intracellular ATP Levels: Cellular Adenosine triphosphate (ATP) wasdetermined with an assay based on a reaction betweenATP+D-luciferin+oxygen catalyzed by luciferase to yieldOxyluciferin+AMP+PPi+CO₂+light. The emitted light is proportional to theamount of ATP present. Rather than a “flash” type signal, which has avery short half-life, this assay utilizes a proprietary “glow”technology that extends the signal half-life to 5 hr. In addition, aunique cell lysis reagent inhibits endogenous ATPases and thereforestabilizes cellular ATP by preventing its degradation to ADP. ATP ispresent in all-living cells and declines rapidly upon cell death. Inaddition, this assay in combination with the MTT assay provides anindicator of mitochondrial activity and energy status of the cell.

At the end of the 24-hr exposure period the medium was removed from thecells and the ATP cell lysis buffer added to each well. Plates wereanalyzed immediately or stored at −20° C. until needed. On the day ofanalysis, the plates were thawed and calibration curve prepared with ATPin the same liquid matrix as samples. ATP was quantified by adding ATPsubstrate solution and then reading luminescence on a Packard FusionLuminescence or equivalent plate reader.

Intracellular Glutathione (GSH) Levels: Intracellular glutathione levelswere determined essentially as described by Griffith (1980) withmodifications. At the end of the exposure period, the medium was removedfrom the cells and metaphosphoric acid (MPA) was added to each well.Plates were then shaken for 5 min at room temperature and stored at −20°C. until needed.

The sample plates were thawed just prior to analysis and centrifugedat >2000×g for at least 2 min. Sample aliquots were removed andtransferred to a clean 96-well plate along with appropriate standardcurve controls. Sample pH was neutralized just prior to analysis andeach well received an aliquot of sodium phosphate reaction buffercontaining EDTA, DTNB, NADPH, and glutathione reductase. The plates weremixed for 15-30 min at room temperature and glutathione content wasdetermined colorimetrically with a Packard Fusion or equivalent readerat 415 nm.

Lipid Peroxidation Measured as 8-Isoprostane (8-ISO): 8-ISO levels weredetermined using an ELISA. 8-ISO is a member of a family of eicosanoidsproduced non-enzymatically by random oxidation of tissue phospholipidsby oxygen radicals. Therefore, an increase in 8-ISO is an indirectmeasure of increased lipid peroxidation (Vacchiano and Tempel, 1994). Atthe end of the exposure period, plates were either analyzed immediatelyor stored at −80° C. until needed for analysis. Color development, whichis indirectly proportional to the amount of 8-ISO present in the sample,was read on a Packard Fusion or equivalent plate reader at 415 nm.

Caspase 3 Procedure: Caspase 3 activity was determined using a caspasesubstrate (DEVD, Asp-Glu-Val-Asp) labeled with a fluorescent molecule,7-Amino-4-methylcoumarin (AMC). Caspase 3 cleaves the tetrapeptidebetween D and AMC, thus releasing the fluorogenic green AMC. Followingthe test article exposure to cells in 96-well plates, medium wasaspirated from plates and PBS added to each well. Plates were stored at−80° C. to lyse cells and store samples until further analysis. On theday of analysis, plates were removed from freezer and thawed. Caspasebuffer with fluorescent substrate was added to each well and incubatedat room temperature for 1 hr. AMC release was measured in aspectrofluorometer at an excitation wavelength of 360 nm and an emissionwavelength of 460 nm. Values are expressed as relative fluorescent units(RFU).

Lipidosis Procedure: After exposure period, the medium in each platewell was removed. Plates were treated with 1 μM Nile Red and incubatedfor 4 hr. Plates were washed and incubated overnight (8-16 hr).Fluorescence was measured using a Packard Fusion or equivalent platereader at 535 nm excitation/580 nm emission.

bDNA Sample Collection and Storage: After 48 hr compound exposure in96-well plates, 50 μl of stock lysis mixture was added to each 100 μlwell. Plates were mixed using a DPC 5 Micromix plate shaker at 37° C.for 15-30 minutes to ensure complete lysis of cells. Cell lysis wasconfirmed using a Nikon inverted microscope. Plates were stored at −80°C. until further analysis.

bDNA Analysis (Day 1): All solutions were prepared fresh on the day ofanalysis. Quantigene® bDNA kit components were brought to roomtemperature prior to use. Chemicals not supplied by Genospectra wereobtained from Sigma-Aldrich.

On the day of analysis, Target Probe Sets were prepared for probe genesets by adding 25 μl of each probe set component (CE, LE, BL) to 425 μlstock lysis solution using the following probe set concentrations: CE(50 fmol/μL), LE (200 fmol/μL), BL (100 fmol/μL).

Cell lysis plates were removed from freezer storage and thawed. Afterthawing, 90 μl of cell lysate (48 hr) was added to each well of theCapture Plate. Next, 10 μl of the appropriate Target Probe Set (PS2) wasadded to each appropriate well of the Capture Plate. Plates were tightlysealed using a plate sealer and incubated at 53° C. for 16-20 hr(overnight).

bDNA Analysis (Day 2): Wash buffer was prepared in ultra-pure waterusing 0.1× Saline-Sodium Citrate (SSC) and 0.03% Lithium Lauryl Sulfate(LLS). Amplifier working solution was prepared by adding 10 μl Amplifierto 10 mL Amplifier Diluent per plate.

Plates were removed from incubator and washed three times with 300 μlwash buffer. Wash buffer was completely removed from plates byaspiration and 100 μl Amplifier working solution was added to each well.Plates were sealed and incubated at 46° C. for 1 hr.

Label Probe working solution was prepared by adding 10 μl Label Probe to10 mL Label Probe Diluent per plate. Upon completion of plate incubationwith Amplifier solution, plates were washed three times with 300 μl washbuffer. Wash buffer was completely removed from plates by aspiration and100 μl Label Probe working solution was added to each well. Plates weresealed and incubated at 46° C. for 1 hr.

Substrate working solution was prepared by adding 30 μl of 10% LithiumLauryl Sulfate to 10 mL Substrate solution per plate. Upon completion ofplate incubation with Label Probe solution, plates were washed threetimes with 300 μl wash buffer. Wash buffer was completely removed fromplates by aspiration and 100 μL Substrate working solution was added toeach well. Plates were sealed and incubated at 47° C. for 30 minutes.Upon completion of last incubation, luminescence was measured using aPackard Fusion or equivalent plate reader with plate heater set to 46°C. Data were collected as relative light units (RLU) and expressed asfold change relative to controls after background subtract.

Dihydro-2′,7′-dichloroflorescin-diacetate (DCFDA) for MonitoringPeroxide Formation: DCFDA (Sigma C-6827) was dissolved in DMSO at aconcentration of 205 mM. The stock was diluted to 50 μM in phosphatebuffered saline. Cells were preloaded with DCFDA for 1 hour prior todosing with test compounds. Positive control wells were dosed withfreshly prepared 300 μM t-Butyl hydroperoxide (tBHP). After compoundexposure, the plates were washed with phosphate buffered saline and readat 485/530 in a Packard Fusion plate reader. Results were corrected foroutliers (+/−2 s.d.) and expressed as fold increase over control values(n=7 per dose).

Solubility: The test compounds were prepared in DMSO and the appropriateamounts were then added to complete medium containing 10% bovine serumand 10% calf serum at 37° C. The samples were evaluated using a lightscattering technique and a Nephaloskan instrument. A reading that wasgreater than or equal to 3 times background was considered the limit ofsolubility.

Assay Calculations

Cell Proliferation: After incubation, the test compound was removed andTriton X-100 solution containing propidium iodide is added. The platewas incubated and read on a fluorescent plate reader. Data was collectedas relative fluorescent units (RFU). The blank was subtracted from thefinal well RFU. It represents the average of the first and last columnswhich contain no cells on that particular plate. The controls areuntreated cells allowed to grow on the same plate. The compounds arerepresented by several separate RFU data points for each of the testedconcentrations. The percent change relative to controls was calculatedby dividing the average RFU by the control cell RFU and multiplying by100.

Bromodexoyurine (BrDU) Incorporation: After incubation, the testcompound/BrDU solution was removed and cells were fixed. A solutioncontaining mouse monoclonal anti-BrDU (FITC) was added and fluorescencewas measured. Data were collected as relative fluorescent units (RFU).The blank was subtracted from the final well RFU. It represents theaverage of the first and last columns which contain no cells on thatparticular plate. The controls are untreated cells allowed to grow onthe same plate. The compounds are represented by several separate RFUdata points for each of the tested concentrations. The percent changerelative to controls was calculated by dividing the average RFU by thecontrol cell RFU and multiplying by 100.

Membrane Leakage (α-Glutathione S-transferase and/or Adenylate Kinase):Leakage of Adenylate Kinase or α-GST from the cell into the culturemedium was determined by collecting the culture medium at the end of theexposure period. Thus, the values measured represent total enzymeleakage lost over the exposure period. The control for 100% dead ormaximum enzyme release was based on cells treated with 1 mM digitonin atthe time of dosing. Percent dead cells relative to digitonin treatedcells was determined and then subtracted from 100% to yield the percentlive cells.

MTT Reductase Activity: At the end of the 24-hr exposure period theculture medium was removed and the remaining attached cells were assayedfor their ability to reduce MTT. Viable cells will have the greatestamount of MTT reduction and hence the highest absorbance values. Percentcontrol values were determined by dividing the meanabsorbance/fluorescence of the treatment group by the mean absorbance ofthe control group and multiplying by 100.

Intracellular ATP Levels: ATP levels in treated cells could bedetermined by using the regression coefficients obtained from the linearregression analysis of the calibration curve. Thus, values could beexpressed as pmoles ATP/million cells. Background corrected luminescencewas used to determine percent change relative to controls by dividingtreated values by control values and multiplying by 100.

Intracellular Glutathione (GSH) Levels: The assay is based on theconcept that all GSH is oxidized to GSSG by DTNB reagent. Two moleculesof GSH are required to make one molecule of GSSG. Total GSH wasdetermined by reducing GSSG to 2GSH with glutathione reductase. Astandard curve was prepared with oxidized glutathione (GSSG) over arange of concentrations. These concentrations are then converted toglutathione equivalents (GSX) essentially by multiplying the GSSGstandard concentrations by 2. The amount of GSX expressed as pmoles/wellwas determined using the standard curve and regression analysis. Thefinal results can be expressed as pmoles GSX/mL by dividing total GSX by0.05 mL, as a percent of control, or as total GSX (pmoles/well).

Lipid Peroxidation Measured as 8-Isoprostane (8-ISO): Backgroundabsorbance produced from Ellman's reagent is subtracted from all wells.Non-specific binding is subtracted from the maximum binding wells togive a corrected maximum binding expressed as Bo. The percent of bound(B) relative to maximum binding capacity (Bo) for all unknown samplesand for standards was determined an expressed as (% B/Bo). The % B/Bofor standards was plotted against the log of 8-ISO added to yield thefinal standard curve. This curve was used to convert % B/Bo to pg8-ISO/mL of sample.

Caspase Activity: After sample plates were completely thawed, thecaspase substrate buffer mix was added to each plate. Plates wereincubated at room temperature for 1 hr, shielded from light. Plates wereread using a in a spectrofluorometer at an excitation wavelength of 360nm and an emission wavelength of 460 nm. Values were expressed asrelative fluorescent units (RFU).

Lipidosis: Nile red incorporation was normalized to cell number andvalues were expressed as fold change relative to controls. Values arenot considered significant unless >2-fold increase over controls.

Dihydro-2′,7′-dichloroflorescin-diacetate (DCFDA) for MonitoringPeroxide Formation: Plates were read at 485/530 in a Packard Fusionplate reader or equivalent. Results were corrected for outliers (+/−2s.d.) and expressed as fold increase over control values (n=7 per dose).

Criteria for the Acceptability and Interpretation of Assays

Assay Acceptance Criteria: The α-GST standard calibration curve fitshould have an r-squared value ≧0.95. The values for unknown samplesmust fall within the calibration curve. The α-GST standard O.D. valuesand negative control O.D. values should be within 15% of historicalvalues. Values within a dose group that vary from the mean by more than±1.4 standard deviations were omitted from the final mean calculation.

Data obtained from the propidium iodide assays were not associated witha calibration or standard curve. Control cells (high fluorescencevalues) and digitonin controls (low fluorescence values) were used asgeneral indicators of assay performance. Values within a dose group thatvaried from the mean by more than ±2 standard deviations were omittedfrom the final mean calculation. For the CyQUANT® Cell ProliferationAssay, the calibration curve for cell number should have an r-squaredvalue ≧0.92. The values of unknown samples must fall within thecalibration curve. It is not possible to dilute samples once theCyQUANT® reagents have been added. Therefore, in those instances wherethe unknown value was outside the calibration curve a value may beassigned, but it should be identified with an asterisk in theappropriate figure or table and considered an estimate of cell number.Values within a dose group that vary from the mean by more than ±2standard deviations were omitted from the final mean calculation.

Data obtained from the MTT assays were not associated with a calibrationor standard curve. Control cells (high absorbance values) and digitonincontrols (low absorbance values) were used as general indicators ofassay performance. Values within a dose group that varied from the meanby more than ±2 standard deviations were omitted from the final meancalculation.

ATP values can be reported either as a percent changes relative tocontrols or as pmol of ATP per million cells. Relative changecalculations are independent of a standard or calibration curve.Negative controls (medium only) and cells without drug and positivecontrols (cells with digitonin) are always included as indicators ofassay function. If the results are reported as an actual amount of ATPper million cells then a calibration curve must be prepared. The slope,intercept, correlation coefficient, and raw lumin±escence values in eachstandard curve are compared to historical values for consistency.Correlation coefficients (r-squared) must be >0.97 in order for thecurve to be considered valid. Unknown samples, within an exposure group,with luminescence values that vary from the mean by more than 2 standarddeviations were omitted from the final mean calculation.

Total GSH is reported as pmols/well at each exposure concentration.Standard curves are prepared fresh for each set of analyses and theslope and intercepts monitored for changes in response and sensitivity.All standard curve data are maintained and routinely evaluated fortrends and for historical comparison. Correlation coefficients mustbe >0.97 in order for the regression analysis to be considered valid forpredicting the concentration of GSH in unknown samples.

8-Isoprostane is a measure of membrane specific lipid peroxidation. Theassay is an ELISA format and values are expressed as picograms of8-isoprostane/mL. Colorimetric values are converted to units of8-isoprostane with regression coefficients obtained from standard curvesand regression analysis. The slope, intercept, correlation coefficient,and standard response are maintained and evaluated historically as anindication of assay performance. Correlation coefficients must be >0.97in order for the regression analysis to be considered valid forpredicting the concentration of 8-isoprostane in unknown samples.

Key to Interpreting the Tox-Panel Data

The Tox-Panel assays were designed to provide information that could beused for the following: (1) to prioritize compounds based on a parameterof toxicity such as the exposure concentration that produces a halfmaximal response in a given assay (TC₅₀); (2) to obtain toxicity datarelative to a reference compound of the same or similar drug class: forexample, ketoconazole might be used as a reference compound whendeveloping new azole antifungals; (3) to provide clues as to potentialsubcellular targets of toxicity; and (4) to provide a link between theexposure concentration that produces toxicity in vitro and the plasmaconcentration that first produces toxicity in vivo.

Example of General Toxicity Data: Referring now to FIG. 62, Rotenone isan extremely potent inhibitor of mitochondrial oxidativephosphorylation. Based on this mechanism of action, Rotenone isconsidered to be a very potent cytotoxic agent. In FIG. 62, the rathepatoma cell line (H4IIE) was exposed to varying concentrations ofrotenone. This example assumes that rotenone is a new drug candidatethat has never been screened for toxicity. Rotenone was evaluated in theTox-Panel, and the information obtained is shown in FIG. 62. After a 6hr exposure, the upper left panel of FIG. 62 shows no cell death asdetermined by leakage of alpha-GST into the medium. If this were theonly assay used to access toxicity, rotenone would be considerednon-toxic.

In the upper right panel of FIG. 62, rotenone caused a reduction in cellnumber. These data alone are unclear because the reduction in cellnumber could be due to cell death or to a reduction in the rate of cellproliferation. When the data in the two upper panels of FIG. 62 arecombined, it becomes clear that the reduction in cell number is not dueto cell death and therefore must be due to a reduction in the rate ofcell proliferation. In the lower left panel of FIG. 62, two markers ofmitochondrial viability are shown, ATP and reduction of MTT. There is adramatic exposure concentration dependent reduction in both ATP and MTT,with ATP being the most sensitive.

When all parameters are plotted together in the lower right panel ofFIG. 62, a more complete profile of rotenone toxicity is seen. Rotenonewas not acutely cytotoxic over the exposure period evaluated (6 hr);however, there were significant effects on mitochondrial function thatoccurred at low exposure concentrations. Thus, this compound has asignificant effect on cellular ATP and this is most likely due to directeffects on mitochondrial function. The slowed rate of proliferation wasdue to reduced rate of proliferation caused by a reduction in cellularATP.

Importance of Oxidative Stress Data: Tissue damage due to production ofreactive oxygen species (ROS) occurs when highly reactive chemicalspecies with unpaired electrons are generated both endogenously and bymetabolism of parent chemicals. The most biologically significant freeradical species are superoxide free radical anion (O₂), hydroxyl radical(OH), and hydrogen peroxide (H₂O₂).

The cellular targets of these ROS are proteins, phospholipids (produceshighly reactive aldehyde molecules), and DNA. The result of theseinteractions is membrane damage, enzyme malfunction, and hydroxylationof DNA, which can lead to mutagenesis.

Oxidative stress can also occur when a chemical is either a directelectrophile or is metabolized to an electrophilic entity. Electrophilescan produce oxidative damage indirectly by depleting cellularantioxidants such as glutathione (GSH) and vitamin E. Once depleted thecell would be considerably more susceptible to oxidant damage fromendogenously produced ROS.

The potential clinical consequences of increased oxidative stressunderscore the importance of evaluating changes in cellular oxidativestress using key biomarkers. The antioxidant defense mechanism ofhealthy cells can be divided into two primary categories: 1) Enzymaticantioxidant systems which would include superoxide dismutase, catalase,peroxidases, and glutathione reductase, and 2) Non-enzymatic oxidantssuch as glutathione, vitamin E and vitamin A. Changes in ATP can also beindicative of oxidative or metabolic stress.

The toxicity panel of the presently disclosed and claimed inventiveconcept(s) provides information on ATP, GSH/GSSG, and membrane lipidperoxidation. In addition, the DCFDA assay for measuring H₂O₂ is alsoavailable.

It is important to remember that oxidative stress is a normal part ofcell death, and if these biomarkers correspond with changes in cellnumber or death, they are the result, not the cause, of the toxicity.Therefore, the most significant mechanistic information is obtained whenATP, GSH, or lipid peroxidation changes occur in a concentrationdependant manner prior to changes in the general cell health biomarkers.When this profile is obtained the information strongly suggests anoxidative mechanism that would eventually lead to significantcytotoxicity.

Example of Oxidative Stress: The left panel of FIG. 63 shows only modesteffects on the general health biomarkers. MTT and AB are reduced, butonly at the highest exposure concentrations. There was essentially nochange in ATP levels at any exposure concentration. In contrast, theright panel of FIG. 63 shows a dramatic concentration dependentreduction in cellular GSH that occurred in the absence of measurablechanges in cell number or cell death. In addition, there was aconcomitant increase in membrane specific lipid peroxidation as measuredby increases in 8-isoprostane. These data indicate that the compoundtested causes significant changes in the cells oxidative state resultingin an increase of lipid peroxidation. These events would in allprobability lead to significant cytotoxicity. This does not mean thecompound above should be immediately dropped from further development.It does however provide a subcellular target and measurable biomarker ofeffect that can be monitored in vivo. If the plasma concentrations ofthe putative drug remain below 20 μM, it is possible that this compoundwould not produce harmful effects under therapeutic conditions. Themechanism of oxidative stress discovered in early screening couldprovide a cautionary flag that could be used to improve preclinicalanimal evaluations as well as human clinical evaluations. Idiosyncratichepatotoxicity associated with many drugs may be linked to subtlealterations in key biochemical systems that control the metabolic andoxidative state of the cell.

Importance of Apoptosis Data: Apoptosis is a mode of cell death by whicha cell can control its own fate. Apoptotic processes occur indevelopment, differentiation, tumor deletion, and in response toexogenous stimuli. The morphologic and biochemical changes associatedwith apoptosis have been well described. There are multiple pathwaysthat can initiate the process of apoptosis. One well characterized andcommitted step is activation of caspase 3. Therefore, caspase 3activation has been included in the general health panel of assays as amarker for initiation of apoptosis. One way of assessing thesignificance of caspase 3 stimulation in vitro is to compare the testcompounds to compounds that are capable of inducing apoptosis in vivo.Internal controls for apoptosis utilized herein include paclitaxel,camptothecin, and staurosporine. Significant increases in caspase 3induction should be seen at exposures that produce cytotoxicity. Thesedata can be seen in FIGS. 64, 64A, and 64B. A key factor when evaluatingthese compounds is the exposure concentration that produced a maximalinduction and the magnitude of the response.

The apoptosis data is expressed in Table 2 as a ratio of the magnitudeof response versus the exposure concentration that produced theresponse. The key to the maximal response data can be found below Table2. By comparing the data in Table 2 for unknown compounds to thepositive controls listed at the bottom of Table 2 and presented ingraphic form below, the relative potency of each compound in Table 2 canbe assessed.

Interpretation of Negative Cytotoxicity Data

In instances where no significant effects are detected in the Tox-Panel,it is important to evaluate all possible experimental or artifactualreasons for this result. For example, toxicity cannot be evaluated if acompound never comes in contact with the test system. Therefore, eventssuch as solubility, protein binding, efflux via membrane transporters,and metabolism must be taken into account. Many of these issues havebeen evaluated in the current Tox-Panel system.

Solubility of the test compounds is evaluated in the dosing medium at37° C. on the day of dosing, and these data are shown in Table 3.

The capacity of the rat hepatoma cell line (H4IIE) to metabolizexenobiotics has been evaluated by determining the constitutive activityof several key cytochrome P450 enzymes. This work has shown theexistence of CYP1A, CYP2B, CYP2C, and CYP3A activity in the H4IIE cells.In addition, there is both glucuronide and glutathione conjugationcapability.

P-glycoprotein (PgP) transport proteins are expressed in the bilecanalicular membrane of normal liver. However, in neoplastic cells it isnot uncommon to have expression of this transporter system in the plasmamembrane. The H4IIE cells have a very active efflux pump activity in theplasma membrane. As a result, compounds that are substrates for thistransporter may not accumulate inside the cell. The existence of thesetransporters was evaluated with the Calcein-AM assay and with knownsubstrates of PgP. The results indicate that these cells havesignificant efflux pump activity. Therefore, compounds that shownegative toxicity should be evaluated as potential substrates of PgP andcompared to data shown in the PgP table of this example.

Example 2 In Vitro Metabolic and Toxicity Screening of Compounds in Ratand Dog Hepatocytes: Species-Selector Analysis

Test compounds were received and processed through the Species-Selectorassay to identify potential differences in metabolic products andtoxicity profiles in the rat as compared to the dog.

The Species Selector Assay was performed as follows:

Step 1: Analysis of metabolic activation: Species-specific formation ofelectrophiles was determined for all 10 compounds using isolated hepaticmicrosomes from dog and rat. Five compounds (COMP A, COMP B, COMP C,COMP D, and COMP E) that induced a differential response between thespecies were selected for metabolic stability and profiling.

Step 2: Analysis of metabolic stability: Metabolic stability for each ofthe five selected compounds was determined by measuring the metabolismof parent compound by LC/MS after incubation in isolated dog and rathepatic microsomes.

Step 3: Analysis of metabolic profiles: The metabolic ‘fingerprint’ foreach of the five compounds from Step 1 was obtained and compared toidentify potential differences. LC/MS analysis of microsome reactionproducts was performed and the resulting chromatographic profilescompared. Two compounds (COMP B and COMP D) that displayed significantdifferences in their metabolite profiles were selected for toxicityprofiling.

Step 4: Toxicity profiling in primary hepatocytes: Toxicity profileswere obtained for COMP B and COMP D in primary hepatocytes from dog andrat using a panel of 9 biochemical markers for cell health.

Results Summary

New drug candidates tested are typically evaluated in rat and dog safetystudies to satisfy regulatory requirements for preparation of an IND. Insome instances a test compound may be considered safe in once specieswhile producing significant toxicity in the other. The ability toidentify and understand the mechanisms underlying species-specifictoxicity is essential for the efficient development of new drugs.

In many cases the reason for the species difference in toxicity is dueto either the rate of metabolism, metabolic activation (defined as theformation of electrophilic intermediates), and the formation of stablebut more toxic metabolites. The ability to identify potential speciesspecific issues prior to performing in vivo studies would savedevelopment time, reduce expense, reduce animal usage, and provide keyinformation that can be used to design more effective and robust animalstudies.

The Species-Selector screen of the presently disclosed and claimedinventive concept(s) is a multi-step process that involves a series ofassays designed to evaluate metabolic activation, rates of metabolism,qualitative differences in the type of metabolite formed, and cell basedtoxicity across each test species.

In the first experiments, each compound was evaluated for potentialmetabolic activation. This assay measures cytochrome P450 (CYP)dependent formation of highly reactive electrophilic intermediates. Theassay also identifies compounds that may have intrinsic electrophilicdomains (e.g., the assay response appears independent of CYP activity).Each of the ten test compounds were evaluated for the formation ofelectrophilic intermediates in rat and dog induced microsomes (FIG. 65).By comparing the two bar graphs for rat and dog, it is clear thatCompounds 17, 18, 29, 30, 39 and 40 had the most pronounced speciesdifferences in this assay.

Metabolic stability was determined for Compounds 17, 18, 29, 30, and 40.The rate of compound metabolism in rat and dog microsomes showed thegreatest species related differences with Compounds 29, 30, and 40(Table 6 and FIG. 66). Compound 17 showed good metabolic stability inrat with a slightly higher rat of metabolism in dog (FIG. 66). Compound18 showed moderate stability in both species (FIG. 66). In terms ofmetabolic stability, Compounds 29 and 40 were metabolized at a higherrate in rat (FIG. 66), while Compound 30 showed the highest rate ofmetabolism in dog (FIG. 66).

Toxicity can be due to the type and quantity of metabolites formed. Aqualitative analysis of the metabolite profiles for Compounds 18, 29,30, and 40 can be seen in FIGS. 67-71. The metabolite profiles forCompound 17 were similar for both rat and dog (FIG. 67). The metaboliteprofile for Compound 18 shows considerably more metabolites are producedin rat than in dog (FIG. 68). The metabolite profile for Compound 29 wassimilar in both species although there may be some differences inrelative abundance (FIG. 69). The metabolite profile for Compound 30showed more metabolites in dog than in rat (FIG. 70).

Results from the metabolic activation assay combined with those from themetabolic stability assay and metabolite profiling were used to selecttwo compounds for in vitro toxicity evaluation with primary hepatocytesfrom rat and dog. Compound 18 produced a P450 dependent metabolicactivation in rat microsomes. Although there was depletion of GSH in dogmicrosomes this did not appear to be dependent on P450 (FIG. 65). Therewere no significant differences in metabolic stability between the twospecies (FIG. 66); however the compound was more extensively metabolizedin rat with multiple metabolites observed as compared to dog where asingle metabolite was measured.

In the cell based cytotoxicity evaluation Compound 18 was more acutelytoxic in dog than rat primary hepatocytes based on the cell viabilitymarker GST (FIGS. 72 and 74). The formation of membrane lipid peroxideswas also much more pronounced in dog than in rat (FIGS. 72 and 74 middlegraph) as measured by 8-isoprostane. Compound 18 should be evaluated ata shorter exposure time in order to identify the most sensitivesubcellular targets. Compound 30 did not produce an acute toxicity ineither species (FIGS. 73 and 75). There was a more pronounced depletionof GSH in rat which appears to be independent of P450.

Taken together these data indicate that Compound 18 would be more toxicin dog while the parent form of Compound 30 may be less toxic in dog dueto more rapid rates of metabolism and similar metabolic profiles betweenthe two species. However, Compound 30 is rapidly metabolized to an M+16metabolite. If this metabolite has inherent toxicity it may become moreapparent under in vivo dosing scenarios that could produce significantblood levels of this metabolite. Compound 30 metabolism should beevaluated in human microsomes to determine if metabolism in the dog isrelevant to the human situation. These data would be valuable indetermining the most relevant second species for toxicity studies.

Species specific toxicity can be due to metabolic stability (change inhalf-life or exposure), formation of reactive metabolites, and speciesspecific targets of toxicity or a combination of these. In order toassess these possibilities all compounds should ideally be compared inthe cell based toxicity screens.

Results

A. Step 1—Metabolic Activation

Test compounds were incubated in a phosphate-buffered solutioncontaining reduced glutathione (GSH) and microsomes fromPhenobarbital/β-Naphthoflavone induced rats. Reactions were initiated byaddition of NADPH, and the disappearance of GSH was monitored using afluorescent probe. Acetaminophen (APAP) was used as the positive controlfor metabolic activation and production of reactive intermediates.Reactions containing the P450 suicide inhibitor Aminobenzotriazole (ABT)and reactions minus NADPH were used as negative controls (see Table 5and FIG. 65).

Analysis of Metabolic Activation Data and Selection of Compounds forMetabolic Stability and Profiling: The metabolic activation results foreach compound were examined, and the five compounds (COMP A, COMP B,COMP C, COMP D and COMP E) demonstrating the greatest difference inactivation between the two species were selected for subsequentanalysis.

B. Step 2—Metabolic Stability

Metabolic stability was conducted using pooled microsomes form induced(BNF/PB) male rat (Sprague-Dawley) animals and induced (BNF/PB) male dog(Beagle) animals. The test compounds were incubated for 30 min at 37° C.at concentrations of 1 μM. Subsequent LC/MS analysis measureddisappearance of the parent molecule. The data are expressed as percentof parent remaining. Positive controls for highly metabolized compoundswere included for comparison. See Table 6 and FIG. 66.

C. Step 3—Metabolic Profiling

Samples from 0 and 60 minute microsomal incubations with 50 μM compoundwere analyzed by LC/MS on a Waters Alliance 2795 LC coupled to aMicromass Quattro micro MS (FIGS. 67-71). The first MS experiment wasperformed with ESI+/− ionization in full scan mode to observe the entirerange of compounds present in each sample. The Total Ion Chromatograms(TIC) obtained for each 50 μM sample are displayed as the first trace ineach of the bottom graphs of FIGS. 67-71. While these traces representtotal ion chromatograms, further insights into the number andcomposition of the metabolites can be obtained from ion specific scans,probing for typically expected species such as addition of one or moreoxygens to the parent molecule or mass. To observe potentialmetabolites, a second MS experiment was performed in Selected IonRecording mode to enhance signal to noise for the metabolites ofinterest. Ion chromatograms from blank matrix samples (withoutcompound), zero time incubations, and 60 minute incubations weregenerated to confirm the metabolic profiles for each species. Thosespecies-specific profiles were then compared to present qualitativedifferences.

Analysis of Metabolic Stability and Profiling Data and Selection ofCompounds for Toxicity Screening: The metabolic stability data revealedthe metabolism (>40%) of COMP B, COMP C, COMP D and COMP E parentcompound in reactions with rat microsomes, and metabolism (>35%) of COMPA, COMP B, COMP C, and COMP D parent compound in reactions with dogmicrosomes. COMP C and COMP E parent compound were metabolized to agreater extent in rat vs dog microsomes after 30 minutes, whereas COMP Aand COMP D were metabolized to a greater extent in dog vs rat microsomesafter 30 minutes. Similar amounts of COMP B parent (50%) remained after30 minutes incubation in either species.

The metabolites produced in each species were examined by LC-MS analysisof microsomal reaction products. FIGS. 67-71 show the total ionchromatograms (top graphs on each page and top trace in bottom graphs)and selected ion chromatographs (bottom two graphs on each page)obtained for each compound. Similar profiles were observed in the totaland selected ion chromatograms for masses corresponding to the parentmass +16 (single oxygen) and parent mass +32 (two oxygens) for COMP Aand COMP E compounds in rat as compared to dog (FIGS. 67 and 71).

Differences were observed in the total and selected ion chromatogramsfor compounds COMP B, COMP C and COMP D (FIGS. 68-70) in reactions withrat versus dog microsomes. For COMP B, multiple mono-oxygenated massesof the parent are observed at various retention times in the rat ascompared to a single mono-oxygenated form in the dog. Threemono-oxygenated forms are seen in both rat and dog for the COMP C (M+16)compound but the relative abundance of two of these forms (at retentiontimes between 4 and 4.5 minutes) are significantly different.Metabolites produced from COMP D showed marked differences when the M+16and M+32 chromatograms are compared between rat and dog. A singleoxygenated species is seen at a retention time of 3.69 minutes and thision is greatly increased in reactions with dog versus rat microsomes.There are additional differences between the M+32 chromatograms for thiscompound.

Based on the stability and profiling results, COMP B and COMP D werechosen for toxicity profiling. COMP B was chosen because it showedsimilar metabolism rates between rat and dog, but the profiles for themetabolites produced were significantly different. COMP D was chosenbecause differences were seen in both stability and profilingexperiments.

D. Step 4—Toxicity Screening in Primary Hepatocytes

Two compounds displaying species-specific differences in metabolicprofiles were selected for toxicity analysis in primary rat(Sprague-Dawley) and dog (Beagle) hepatocytes. Hepatocytes were seededby CellzDirect into 96-well plates, shipped overnight to the inventor,and maintained in medium containing 20% bovine serum. Following anequilibration period of 24 hr, the cells were treated with the P450inducers (50 μM Phenobarbital (PB)+15 μM beta-Naphthoflavone (BNF)).After the 24 hr induction period, cells were treated with test compoundsat concentrations of 0, 1, 5, 10, 20, 50, 100, and 300 μM for 48 hr at37° C. in 5% CO₂. The cell supernatant or the cells themselves wereharvested for biochemical analysis. General cytotoxicity was evaluatedby monitoring membrane integrity, mitochondrial function, cell number,oxidative stress, and apoptosis. In addition to monitoring biochemicalchanges essential for cell health, solubility was also assessed. Themeans of each exposure group (n=3-7) were calculated for each assayperformed.

The response of each assay must be compared to the change in cell numberin order to ascertain whether the change was directly due to the testcompound or the result of fewer cells. Thus, for correct interpretationof results, all data must be visually normalized to cell number withinthe graphs. For example, if a decrease in the mitochondrial markerstrack with loss of cell number, then the toxicity is likely notmitochondrial-specific but related to cell number. If the mitochondrialmarkers are decreased without a reduction in cell number, then thetoxicity is likely specific to the mitochondria. In another example, iftotal glutathione (GSH) is decreased in the absence of decreased cellnumber, then the toxicity is likely glutathione related. Cell number andcell viability are key parameters for correct interpretation of results.

The results are summarized in Tables 7-9 and in FIGS. 72-75. Table 7compares the test compound TC₅₀ values for each assay endpoint using ratand dog primary hepatocytes. Test compounds are rank-ordered from mostto least toxic based on their TC₅₀ values and overall shape of theresponse curves. Assay response is continually monitored to assurereliable results. Table 8 summarizes oxidative stress and apoptosisdata. Table 9 summarizes the solubility data.

Materials and Methods

Experimental Protocol for Toxicity Screening: The test compounds wereprovided by the sponsor and shipped to the inventor for testing. Thecompound was stored at 4° C. until needed. Dosing solutions wereprepared in complete culture medium. Primary hepatocytes were used asthe test system. Cells were received from CellzDirect, shipping mediumwas replaced with culture medium, and cells were given a 24 hrequilibration period. Following an equilibration period of 24 hr, thecells were treated with the P450 inducers (50 μM Phenobarbital (PB)+15μM beta-Naphthoflavone (BNF)). After the 24 hr induction period, cellswere treated with test compounds at concentrations of 0, 1, 5, 10, 20,50, 100, and 300 μM. Solubility was determined by Nephalometrytechniques immediately after dosing and prior to harvesting the cells at48 hr. Following the exposure period, the cells or their supernatant(culture medium) were analyzed for changes in cell proliferation,membrane leakage, mitochondrial function, oxidative stress, andapoptosis. The resultant exposure concentration response curves weregraphed and analyzed for determining the concentration that produced ahalf maximal response or TC₅₀.

Test and Control Articles: The test compounds were received dry or as aliquid and were used to prepare 20 mM stock solutions in DMSO. Thisstock was diluted in DMSO to prepare 0.2 mM stock solutions. Both the 20mM and 0.2 mM stocks were used to prepare dosing solutions of 0, 1, 5,10, 20, 50, 100, and 300 μM in culture medium. The final concentrationof DMSO in the 0-100 μM solutions was 0.5% and at the 300 μM solutionDMSO was 1.5%. The final dosing solutions in medium were prepared on theday prior to dosing. The solutions were wrapped in foil or shielded fromlight and stored at 4° C. until needed.

All experiments used dimethylsulfoxide (DMSO) as the test articlesolvent and the negative (solvent) control.

Negative controls of medium plus DMSO (0.5%) were included with andwithout cells. A positive control for complete cell death received 1 mMdigitonin in medium on the day of dosing.

Reagents and Solutions: All chemicals used were reagent grade or better.

Test Systems: Rat and Dog Primary Hepatocytes: Primary hepatocytes werepurchased from CellzDirect and received plated and sandwiched withMatrigel overlay. The culture medium used for these cells was Williams Emedium with supplements and 20% bovine serum. Certified bovine serum wasfrom In vitrogen.

Description of Experimental Setup and Biochemical Assays:Collagen-coated 96-well plates were seeded with 100,000 primaryhepatocytes by CellzDirect, monitored overnight for attachment, andshipped the following day. Upon arrival, shipping medium was replacedwith culture medium. Cells were dosed the following day after 24 hrequilibration time. Test compounds in medium were added to the platesafter the equilibration period.

During method development experiments, it was determined that the 48 hrcell growth period for NRK cells allows cells to move into a stablegrowth phase prior to treatment. For rat primary hepatocytes, the 4 hrplating period allows cells to attach to the collagen base prior totreatment.

In previous studies, the effect of DMSO on cell number, MTT, and

-GST was evaluated at DMSO concentrations ranging from 0.05 to 4%. Thesestudies showed no effects on any of the endpoints tested atconcentrations below 2%. Finally, the ability of DMSO to enhance celluptake and hence toxicity of a compound was also evaluated. Nosignificant differences in toxicity were detected when a broad range ofketoconazole and amphotericin concentrations were tested at final DMSOconcentrations of 0%, 0.5% and 1.5%.

Biochemical assays of cell number (i.e., cell proliferation), membraneleakage, tetrazolium dye reduction (MTT), intracellular ATP andGlutathione (GSH) levels, lipid peroxidation and caspase-3 wereperformed as described above in Example 1.

Solubility: The test compounds were prepared in DMSO and the appropriateamounts were then added to complete medium containing 10% bovine serumand 10% calf serum at 37° C. The samples were evaluated using a lightscattering technique and a Nephaloskan instrument. A reading that wasgreater than or equal to 3 times background was considered the limit ofsolubility.

Metabolic Activation: Test compounds were incubated in aphosphate-buffered solution containing reduced glutathione (GSH) andmicrosomes from Phenobarbital/β-Naphthoflavone induced rats. Reactionwas initiated by addition of NADPH, and disappearance of GSH wasmonitored using a fluorescent probe. Acetaminophen (APAP) was used asthe positive control for metabolic activation and production of reactiveintermediates. Reactions containing the P450 suicide inhibitorAminobenzotriazole (ABT) and reactions minus NADPH were used as negativecontrols. Assays were performed in 96-well micro plates and fluorescencewas read using a Packard FluoroCount reader with proper emission andexcitation filters.

Metabolic Stability and Profiling by mass spectrometry: Metabolicstability was conducted using pooled male dog (beagle) microsomes andmale rat (Sprague-Dawley) microsomes from induced(beta-Naphthoflavone/Phenobarbital, BNF/PB) animals. The test compoundswere incubated for 30 min at 37° C. at concentrations of 1 and 10 μM.Subsequent analysis by mass spectrometry measured disappearance of theparent molecule. The data are expressed as percent of parent remaining.Positive controls for highly metabolized compounds were included forcomparison.

For selected ion monitoring, the 50 μM reaction mixtures are incubatedat 37° C. for 0 or 60 minutes, after which the reaction is quenched withacetonitrile, centrifuged and supernatant collected. Supernatant isconcentrated with nitrogen gas, re-suspended in acetonitrile/H₂O (50/50)and analyzed by LC/MS for metabolite appearance. Reaction mixtureswithout substrate are used for background determination. Duplicateincubations are run at each time-point.

The samples from the microsomal incubations are analyzed by LC/MS on aWaters Alliance 2795 LC coupled to a Micromass Quattro micro MS. Thechromatography is performed on a Waters SymmetryShield RP8 5 um 3.0×150mm column. Solvent A is water with 0.1% formic acid and Solvent B isacetonitrile with 0.07% formic acid. The gradient is programmed from 97%A to 5% A over 6 minutes. The first MS experiment is run with ESI+/−ionization in full scan mode to observe the entire range of compoundspresent in the samples. Additional MS experiments are performed inSelected Ion Recording mode to enhance signal to noise for themetabolites of interest. Ion chromatograms from blank matrix samples(without compound), zero time incubations, and 60 minute incubations aregenerated to confirm the metabolic profiles for each species.

Assay calculations and Criteria for Acceptability and Interpretation ofAssays were performed as described in Example 1, except as describedherein below.

Assay acceptance criteria: Data obtained from the WST-1 assays were notassociated with a calibration or standard curve. Control cells (highfluorescence values) and digitonin controls (low fluorescence values)were used as general indicators of assay performance. Values within adose group that varied from the mean by more than ±2 standard deviationswere omitted from the final mean calculation.

Metabolic Activation: Data was collected from 96-well plates using aPackard FluoroCount reader with the appropriate excitation and emissionwavelengths. Wells containing an assay mixture of reduced GSH,microsomes, NADPH, and fluorescent probe (minus compound) were used ascontrols. Data processing and calculations were performed usingMicrosoft Excel. Background fluorescence was subtracted from control andtreated samples. Percent control values were determined by dividing themean fluorescence value of the treatment group by the mean fluorescencevalue of the control group and multiplying by 100. Bar graph wasprepared using Excel.

Metabolic Stability: The analysis measured disappearance of the parentmolecule. The data were expressed as percent of parent remaining.Positive controls for highly metabolized compounds were included forcomparison.

Example 3 In Vitro Toxicity Screenings of Compounds in Rat Hepatoma(H4IIE) Cell Line, Rat Primary Hepatocyte Cell Line, and Normal RatKidney (NRK) Cell Line: an Anti-Tumor Screen

Three separate projects were carried out in series. The individualprojects are presented in the order in which they were done. The bargraph in the third project summarizes the data showing cell selectivity.

A single compound was submitted for evaluation in a non-GLP generalcytotoxicity screening platform. The compound was submitted, as a liquidand the dosing was based on a series of dilutions recommended by thesponsor. The dosing dilutions used in all experiments were 2000, 1000,500, 100, 50, 10, and 1. The highest dilution represents the lowestexposure concentration and the lowest dilution the highest exposureconcentration. The test compound was acidic in nature and was designedas an antitumor drug.

In the first experiment, the test compound was evaluated for toxicityusing a rat hepatoma cell line. The results shown in the TABLE 10 and inFIGS. 76A-D indicate that the compound was a potent cytotoxic agent. Inorder to separate toxicity related to the acidic nature of the compoundfrom specific effects related to the compound itself, a series ofexperiments were done to evaluate the effects of pH on the test system.This information was related to the pH values of the actual dosingsolutions. The pH values of the 2000, 1000, and 500 dosing solutionswere near 7.0. The next highest exposure (100) dropped the pH to 3.7 avalue considerably lower the test range of 7.5 to 6.0 (FIGS. 77A-B). InFIGS. 76A-D below, nearly all cells were dead at the 500 dilution at 24hr. This cytotoxic effect was not due to low pH as the 500 dilution hada pH of near 7.0 and the cells were stable at this pH. The extremely lowpH values produced by the compound did have negative effects onmitochondrial markers (MTT and ATP) as shown in FIGS. 77A-B. Theoxidative stress markers and apoptotic markers had toxicity profilesconsistent with the loss of cell viability.

In the second experiment, cryopreserved rat primary hepatocytes wereused as the test system. These cells were purchased from XenoTech LLC ofKansas City, Kans. The data are summarized in TABLES 10B and 11B. Theobjective of this experiment was to evaluate the toxicity of theantitumor test compound on cells derived from normal tissues. Theresults of this experiment revealed that the cytotoxic effects of thetest compound at dilutions within physiologic pH were selective fortumor cells. At the highest exposure concentration (dilution of 500)there were no changes in cell number or in acute cell death (FIGS.76E-I) after either 6 or 24 hr exposure. Again, the low pH inherent withthis test material did produce effects on mitochondria as shown in FIGS.77C-F. However, these effects of pH were not observed at pHs of 7.0 andhigher. Thus, the cytotoxicity of the test material must be due to thechemical mixture and not to the physical chemical changes conferred ontothe test system.

The third experiment was done to evaluate the specificity andselectivity of the test compound in another non-tumor cell that wasderived from normal rat kidney tissue. The results are summarized inTABLES 10C and 11C. In this experiment the test compound has no effecton cell number of acute cell death up to the 500 dilution after 6 and 24hr of exposure (FIGS. 76J-O and 77G-J). Significant effects wereobserved on mitochondrial markers at dilutions that dropped pH to 3.7.The bar graph in FIG. 82 summarizes these effects. Both non-tumor celltypes showed no acute cytotoxicity at dilutions that maintained a pHnear 7.0. In contrast the rat tumor cell line was sensitive to the testcompound and cell death occurred at the 500 dilution and could not beattributed to low pH.

FIGS. 78-91 illustrate the positive controls Camptothecin and Rotenonein NRK cells at 6 and 24 hour exposures.

UNIQUE OBSERVATIONS AND COMMENTS RELATED TO TOXICITY: The anti tumordrug known as Comp A was evaluated in a previous study for cytotoxicityusing a rat hepatoma derived cell line. Comp A was cytotoxic to thetumor cells at a dilution of 1:500. This dilution had a pH ofapproximately 7. In addition, there was a significant increase in theactivity of caspase 3, a late stage marker of apoptosis. At higherexposures the pH dropped to levels below 5 and therefore effects on thecells at this extremely acidic pH could not be differentiated fromdesired effects of the drug itself. In the present experiment theobjective was to evaluate Comp A cytotoxicity on non-tumor or cellsderived from normal tissues. For this example, cryopreserved rat primaryhepatocytes were obtained from a well established distributor. Thedilutions of Comp A were the same as those used in the previous example.The lowest exposure concentrations (1:2000, 1:1000, and 1:500) did notproduce acute cytotoxicty in the normal liver cells. There was however,a significant reduction in ATP and MTT indicating some stress onmitochondrial function (FIG. 76, 24 hr). The pH was again maintained atapproximately 7. Again, the higher exposures had pH values well below 5.Experiments designed to evaluate the effects of pH on the cells revealedsignificant effects on mitochondrial function, but not cell death, atpHs below 6.5 (FIG. 77). These data also suggest that low pH can induceapoptosis. Thus, it is clear that a condition of extremely low pH wouldhave direct effects on cell health. However, at the 1:500 exposure levelall cells were viable in the normal liver cell model. This is incontrast to the effects observed in the tumor cell line where allmarkers of cell viability were at maximum reductions at the 1:500dilution. These data indicate that the tumor cell line under conditionsof near physiologic pH is considerably more sensitive to the cytotoxiceffects of Comp A than are the non-tumor cells (see FIG. 82), based oncell number and membrane integrity. The Comp A drug does produce adverseeffects on mitochondrial function, and that occurs in both cell types.

Materials and Methods were conducted as described in Example 1, exceptas described herein below.

Test System: Cell Lines: Rat hepatoma derived H4IIE cells were used asthe test system. The culture medium used for these cells was EaglesMinimum Essential Medium with 10% FBS and 10% calf serum. Certified FBSand calf serum were from InVitrogen.

Experimental setup was the same as in Example 1, except that no DMSO wasused.

Example 4 In Vitro Toxicity Screening of Compound a (ProtectiveAntigen:Lethal Factor) in SK-MEL28, Human Hepatocyte, HUVEC, C32, andNHEM Cells Multiple Cell Screen to Identify Drug Specificity and SpeciesSensitivity

The test compound consisted of two parts mixed in a constant ratio of5:1. The final test mixture consisted of Test mix 1 and Test mix 2,which were received as frozen solutions. The test mixture was tested ina ratio of 5:1 COMP A in five different cell types which included thefollowing: SK-MEL28, human hepatocytes, HUVEC, C32, and NHEM cells.Cells were seeded into 96-well plates and cultured in medium containing10% bovine serum. Following an equilibration period of 48 hr, the cellswere treated with the test compound mix at concentrations of COMP A(μg/mL)=(250:50), (125:25), (25:12.5), (12.5:2.5), (2.5:1.25),(1.25:0.25), and (0.25:0.05) for 24 hr (overnight) at 37° C. in 5% CO₂.Camptothecin and rotenone were included as positive control compoundsbecause they produce measurable effects in all of the assays. Followingan equilibration period of 4 hr, the cells were treated with the testcompound mix at concentrations of COMP A (μg/mL)=(250:50), (125:25),(25:12.5), (12.5:2.5), (2.5:1.25), (1.25:0.25), and (0.25:0.05) for 24hr (overnight) at 37° C. in 5% CO₂.

The cell supernatant or the cells themselves were harvested forbiochemical analysis. General cytotoxicity was evaluated by monitoringmembrane integrity, mitochondrial function, cell proliferation,oxidative stress, and apoptosis. In addition to monitoring biochemicalchanges essential for cell health, interaction with P-glycoprotein (PgP)and solubility were also assessed. The means of each exposure group(n=3-7) were calculated for each assay performed. Intra assay variationwell to well was typically less than 10% with plate to plate variationtypically less than 20%.

The response of each assay must be compared to the change in cell numberin order to ascertain whether the change was directly due to the testcompound or the result of fewer cells. Thus, for correct interpretationof results, all data must be visually normalized to cell number withinthe graphs. For example, if a decrease in the mitochondrial markerstrack with loss of cell number, then the toxicity is likely notmitochondrial-specific but related to cell number. If the mitochondrialmarkers are decreased without a reduction in cell number, then thetoxicity is likely specific to the mitochondria. In another example, iftotal glutathione (GSH) is decreased in the absence of decreased cellnumber, then the toxicity is likely glutathione related. Cell number andcell viability are key parameters for correct interpretation of results.

The results are summarized in Tables 14-17 and in FIGS. 83-102. Table 14compares the test compound TC₅₀ values for each assay endpoint. Testcompounds are rank-ordered from most to least toxic based on their TC₅₀values and overall shape of the response curves. The C_(tox) value, orestimated sustained blood concentration where toxicity would first beexpected to occur in a rat 14-day repeated dose study, is also includedin this table. Negative and positive controls are included with everyrun. Assay response is continually monitored to assure reliable results.Camptothecin and rotenone were included as positive controls for allendpoints, while DMSO at 0.5% in culture medium was included as anegative control. Table 15 summarizes oxidative stress and apoptosisdata, Table 16 summarizes the solubility data, and Table 17 providesinformation on interaction with PgP.

The C_(tox) value was developed using 24 hr in vitro (H4IIE) toxicitydata and was validated by retrospectively evaluating compounds acrossdifferent classes of drugs in 14-day rat studies in which compounds wereadministered on a daily basis and pharmacokinetic data was available.These evaluations showed that the C_(tox) value was an accurateprediction of in vivo toxicity. Thus, C_(tox) values are only determinedfor the 24 hr in vitro results. In this example, C_(tox) values couldnot be estimated due to low toxicity in most of the cell lines.

Summary of toxicity data from MTT and glutathione (GSH) assays acrossall cell types compared with positive cell data from the sponsor. Theexposure concentration in all cases was PA: LF ration of 250:50 μg/mL.In an effort to maintain quality of data between laboratories, data wasrequested from the sponsor on SK-MeI 28 sensitivity. The MTT dataobtained in this assay was essentially identical to the responsereported by the sponsor. FIG. 83 also demonstrates varying degrees ofsensitivity between cell types. Normal rat kidney cells (NRK) and humanhepatocytes were the least sensitive. Note: the NRK cells and ratprimary hepatocytes were tested in a previous project and are includedin this summary graph for comparison purposes. SK-Mel28, NHEM, C32, andthe rat primary hepatocytes were the most sensitive cell types and canbe rank ordered from most to least sensitive as follows: Rat primaryhepatocytes >NHEM=SK-Mel 28>C32>HUVEC>Human hepatocytes>NRK. Two keyfindings can be seen from these data. First human hepatocytes, and aproliferating cell line not derived from tumor tissue were the leastsensitive. Human umbilical vein endothelial cells (HUVEC) were alsoresistant. The melanoma derived cell lines were sensitive to thecytotoxic properties of the test material. Interestingly, rat primaryhepatocytes were the most sensitive. This finding indicates that the ratmay be a sensitive species in terms of liver toxicity. In comparison,human primary hepatocytes were very resistant to toxicity. The exposureconcentrations chosen for this study were high. As a result, many of thecells exhibited maximal effects at the lowest exposure concentration. Anexample can be seen in FIGS. 84A-D. The upper panel represents the highexposure concentration range and shows a maximal effect of about 50%.This is a typical cytostatic response pattern. When the dose range wasextended into the lower concentrations, the full response curve could bedescribed and an IC50 value determined.

As shown in FIGS. 84A-B, initial experiments using a high range of dosesrevealed a flat response at 50% for MTT (red triangles) with no effecton other markers of toxicity. MTT can be used to assess changes in cellnumber or mitochondrial function. The data shown in FIG. 84A reflectchanges in cell number without acute toxicity because ATP levels werenot affected by the test compound. Furthermore, the MTT assay proved tobe a more sensitive marker for cell number than the propidium iodideassay (FIG. 84A, circles). In order to define a dose-responserelationship, the MTT assay was used to monitor change over a lowerexposure range (FIG. 84B). The lower end exposures used were as follows:2.5/0.5, 1.25/0.25, 0.25/0.05, 0.125/0.025, 0.025/0.005, 0.0125/0.0025,0.0025/0.0005 μg/mL COMP A. The IC50 value obtained from these data was0.25/0.05. No effects on GSH (FIG. 84C, circles) or Caspase 3 (FIG. 84D,circles) were detected.

Experiments were also conducted to verify the relationship betweenexposure time and the observed cytostatic effects of the test compound.An additional assay was used to monitor cumulative change in DNAreplication. The assay uses the thymidine analog BrdU to measure S-phaseof cell replication and is considered to be the Gold Standard for cellproliferation. The data in FIGS. 85A-B confirm that an increase inexposure time from 24 to 72 hr increased the cytostatic response(inhibition of cell proliferation) without an increase in acute celldeath (See FIG. 89 for comparison to a known cytostatic drug). The BrdUassay paralleled the MTT and propidium iodide assays. The apparent lowersensitivity of the BrdU assay is a reflection of the experimental designnot the assay. BrdU data represents a cumulative effect from thebeginning of exposure while the other assays are measuring one isremaining at the end of either the 24 hr or 72 hr exposure period. Totalglutathione levels were also reduced in this experiment (FIGS. 85C-D).There was a trend toward increasing activity in caspase-3, a marker forcell apoptosis (FIGS. 85E-F).

In FIGS. 86A-C, the effect of the test compound on human primaryhepatocytes was examined. There were no detectable changes in any of themarkers of cytotoxicity over the 35 hr exposure period.

FIGS. 87A-C depicts the effects of the test material on a humanumbilical vein endothelial cell line. There were no detectable changesmeasured in any of the acute toxicity markers (FIGS. 87A and C). Therewas a significant increase in membrane lipid peroxidation as determinedby 8-isoprostane (FIG. 87B).

The data presented in FIGS. 88A-C and 89A-F are similar to the responseobserved in SK-MeI 28 cells at the high exposure concentrations. MTT wasthe most sensitive marker to cell proliferation. Higher exposuresactually produced a moderate increase in cells with a compensatoryincrease in ATP levels.

In FIGS. 90A-C, MTT was again the most sensitive assay at the highexposure concentrations. ATP increased in what was most likely acompensatory response. Total GSH was reduced at the highest exposures,and there was no cell death as determined by membrane leakage (opensquares).

FIGS. 91A-C and 92A-F represent 24 and 72 hr cytotoxicity data followingcamptothecin exposure. In FIG. 92A, following a 24 hr exposure, thecells respond with a drop in MTT, cell number, and ATP. Cellproliferation as measured by the S-phase marker BrdU was undetectableafter a 24 hr exposure to 5 and 10 μM. These data indicate thatcamptothecin inhibits cell proliferation prior to cell death. Formulti-cell comparison, FIGS. 93A-C, 94A-C, 95A-C, and 96A-C represent 24hr cytotoxicity data following camptothecin exposure in humanhepatocytes, HUVEC cells, C32 cells, and NHEM cells, respectively.

In FIGS. 99A-C, human primary hepatocytes were exposed to rotenone, aninhibitor of oxidative phosphorylation and ATP synthesis bymitochondria. These data provide a good example of ATP depletion andmitochondrial damage prior to acute cell death and loss of cells. Formulti-cell comparison, FIGS. 97A-C and 98A-F represent 24 hr and 72 hrcytotoxicity data following rotenone exposure in SK-MEL28 cells, whileFIGS. 100A-C, 101A-C, and 102A-C represent 24 hr cytotoxicity datafollowing rotenone exposure in HUVEC, C32 and NHEM cells, respectively.

Materials and Methods

All Materials and Methods, including Biochemical assay protocols, assaycalculations, and Criteria for the Acceptability and Interpretation ofAssays/Tox-Panel Data/Negative Cytotoxicity Data, were performed asdescribed in Example 1, with the exceptions of those Materials andMethods described herein below.

Experimental Protocol: The test compounds were shipped on dry ice fortesting. The compounds were stored frozen at −80° C. until needed.Dosing solutions were prepared in complete culture medium. SK-MEL28,human hepatocytes, HUVEC, C32, and NHEM cells were used as the testsystems. Cells were seeded into 96-well plates and allowed toequilibrate for approximately 48 hr. Following the equilibration periodthe cells were exposed to the test compound mix at concentrations ofCOMP A (μg/mL)=(250:50), (125:25), (25:12.5), (12.5:2.5), (2.5:1.25),(1.25:0.25), and (0.25:0.05). Solubility was determined by Nephalometrytechniques immediately after dosing and prior to harvesting the cells at24 hr. Following the exposure period, the cells or their supernatant(culture medium) were analyzed for changes in cell proliferation,membrane leakage, mitochondrial function, oxidative stress, andapoptosis. The resultant exposure concentration response curves weregraphed and analyzed for determining the concentration that produced ahalf maximal response or TC₅₀.

Test and Control Articles: The test compounds were received as frozensolutions of 4.4 mg/mL Protective Antigen (PA) and 4.56 mg/mL LethalFactor (LF) prepared in 5 mM HEPES, pH 7.4. The stock solution were usedto prepare dosing solutions of COMP A (μg/mL) High Dose range=(250:50),(125:25), (25:12.5), (12.5:2.5), (2.5:1.25), (1.25:0.25), and(0.25:0.05) in culture medium. In some experiments a low dose range wasalso included with COMP A ratios of (2.5:0.5), (1.25:0.25), (0.25:0.05),(0.125:0.025), 0.025:0.005), (0.0125:0.0025), 0.0025: 0.0005) μg/mL. Thedosing solutions in medium were prepared on the day of dosing. Thedetails of the preparation and dilutions can be found in the laboratory.A positive control for complete cell death received 1 mM digitonin inmedium on the day of dosing.

Reagents and Solutions: All chemicals used were reagent grade or better.Cell culture supplies were from InVitrogen or Sigma/Aldrich.

Test Systems: SK-MEL28, human primary hepatocytes, HUVEC, C32, and NHEMwere used as the test systems. The cell culture conditions forSK-MeI-28, HUVEC, and C32 cells were provided by the sponsor.

Culturing Conditions:

SK-MeI-28 cells. The cells were grown in RPMI 1640 medium withL-glutamine. The cells were cultured in the presence of penicillin andstreptomycin at concentrations of 1% (v/v) in the final culture medium.Fetal bovine serum (FBS) was added to yield a final concentration of 5%.Serum and antibiotics were obtained from InVitrogen. The cells wereseeded into 96-well culture plates at a density of 10,000 per well andallowed to equilibrate for 48 hr prior to dosing.

HUVEC cells: Normal human umbilical vein endothelial cells: Cells werepurchased from Cambrex. The EGM-2 bullet kit was used for culture. Thecells were grown in 1% fetal bovine serum (FBS). The cells were seededinto 96 well culture plates at a density of 10,000 per well and allowedto equilibrate for 48 hr prior to dosing.

C32 cells: Human amelanotic melanoma cells. Cells were grown in MEM withEarle's salts and L-glutamine. The media was supplemented with 1%penicillin and streptomycin, sodium pyruvate, essential amino acids, and10% fetal bovine serum (FBS). Cells were seeded into 96-well cultureplates and allowed to equilibrate for 48 hr prior to dosing.

NHEM cells: Normal human epidermal neonatal melanocytes. Cells weregrown in media prepared by Cambrex in their MGM-4 bullet kit. The finalserum concentration was 0.5% fetal bovine serum (FBS). These cells haveextremely slow growth characteristics with doubling time ofapproximately 73 hr. These cells were seeded at a density of 12,500 perwell and allowed to equilibrate for one week prior to dosing.

Rat Primary Hepatocytes: Cryopreserved cells were purchased fromXenoTech LLC. (Kansas City, Kans.). The cells were seeded into 96-wellplates at a density of 100,000 per well. The cells were allowed toequilibrate approximately 4 to 6 hr in seeding medium. During this timethe cells formed a monolayer on the plate. After 4 to 6 hr the mediumwas removed and fresh culture medium containing 10% fetal bovine serumand test compound was added back to the wells.

Human Primary Hepatocytes: Cryopreserved cells were purchased fromXenoTech, LLC, (Kansas City, Kans.). Cells were seeded into 96-wellBiocoat plates at a density of 40,000 per well in seeding medium. Thecells were allowed to equilibrate and form adherent monolayer overnight. Following the equilibration period the medium was removed thefresh culture medium containing 10% fetal bovine serum and the testcompound was added back to the wells.

For all cell types, the primary exposure time was 24 hr. In someinstances, the exposure time was increased to 72 hr. When this occurred,both the 24 and 72 hr data were shown and clearly labeled.

Description of Experimental Setup and Biochemical Assays: Flat bottom96-well plates were seeded with at the densities specified above undertest systems. Collagen-coated 96-well plates were seeded with 100,000rat primary hepatocytes 4 to 6 hr prior to dosing. Test compounds inmedium were added to the plates after the equilibration period.

Solubility Assay: The test compounds were prepared in DMSO and theappropriate amounts were then added to complete medium containing 10%bovine serum at 37° C. The samples were evaluated using a lightscattering technique and a Nephaloskan instrument. A reading that wasgreater than or equal to 3 times background was considered the limit ofsolubility.

Thus, in accordance with the presently disclosed and claimed inventiveconcept(s), there has been provided methods of determining a level oftoxicity for a chemical compound, as well as methods of determiningorgan-specific and species-specific toxicities, that fully satisfies theobjectives and advantages set forth hereinabove. Although the presentlydisclosed and claimed inventive concept(s) has been described inconjunction with the specific drawings, experimentation, results andlanguage set forth hereinabove, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the presently disclosed and claimed inventive concept(s).

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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TABLE 1 Summary of General Cytotoxicity (RAT CARDIOMYOCYTES) Cell NumberMemTox MTT ATP Predicted Compound TC₅₀ TC₅₀ TC₅₀ TC₅₀ C_(tox)Name/Number (μM) (μM) (μM) (μM) (μM) CLIENT Compound A CM 1 HR ND >300276 185 NA CLIENT Compound A CM 3 HR ND >300 233 181 NA CLIENT CompoundA CM 6 HR ND >300 187 163 NA CLIENT Compound A CM 24 HR ND >300 64 98 NAADRIAMYCIN CM 1 HR ND >300 >300 >300 NA ADRIAMYCIN CM 3 HR ND >300 41 62NA ADRIAMYCIN CM 6 HR ND 40 18 29 NA ADRIAMYCIN CM 24 HR ND 7 1 6 NAIDARUBICIN CM 24 HR ND <1 <1 2 NA MITOXANTRONE CM 24 HR ND 2 1 2 NADAUNORUBICIN CM 24 HR ND 4 6 9 NA PIRARUBICIN CM 24 HR ND 6 7 7 NAEPIRUBICIN CM 24 HR ND 35 16 19 NA RITONAVIR CM 24 HR ND 83 75 69 NAEFAVIRENZ CM 24 HR ND 69 79 75 NA LOPINAVIR CM 24 HR ND 76 72 72 NADELAVIRDINE CM 24 HR ND 239 252 166 NA ABACAVIR CM 24 HR ND >300 >300165 NA INDINAVIR CM 24 HR ND >300 >300 >300 NA NEVIRAPINE CM 24 HRND >300 >300 >300 NA AZT CM 24 HR ND >300 >300 >300 NA ROTENONE CM 24 HRND 56 33 8 NA CAMPTOTHECIN CM 24 HR 293 >300 >300 >300 NA Summary ofGeneral Cytotoxicity (H4IIE CELLS) Ctox Ranking (μM) - Probability of invivo Effects 1 High 20 21 Moderate 50 51 Low 300 Cell Number MemTox MTTATP Predicted Compound TC₅₀ TC₅₀ TC₅₀ TC₅₀ C_(tox) Name/Number (μM) (μM)(μM) (μM) (μM) CLIENT Compound A H4IIE 24 HR 236 >300 163 164 60ADRIAMYCIN H4IIE 24 HR 18 33 13 7 1 IDARUBICIN H4IIE 24 HR 4 30 3 2 1DAUNORUBICIN H4IIE 24 HR 8 41 14 7 3 PIRARUBICIN H4IIE 24 HR 8 50 16 8 5DOXORUBICIN H4IIE 24 HR 9 16 26 22 7 EPIRUBICIN H4IIE 24 HR 31 37 81 328 MITOXANTRONE H4IIE 24 HR <1 >300 >300 5 11 EFAVIRENZ H4IIE 24 HR 169181 161 162 58 RITONAVIR H4IIE 24 HR >300 >300 219 249 52 DELAVIRDINEH4IIE 24 HR >300 >300 218 257 68 LOPINAVIR H4IIE 24 HR >300 >300 184 19560 ABACAVIR H4IIE 24 HR >300 >300 >300 154 65 INDINAVIR H4IIE 24 HR 300300 300 300 90 NEVIRAPINE H4IIE 24 HR >300 >300 >300 >300 100 AZT H4IIE24 HR >300 >300 >300 >300 ND ROTENONE H4IIE 24 HR 0.04 100 0.07 0.040.03 CAMPTOTHECIN H4IIE 24 HR 4 >300 1 1 0.1 CM = Rat Cardiomyocyte.MemTox = Membrane permeability: AK = Adenylate kinase, GST =α-glutathione S-transferase (membrane leakage). MTT =3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, ATP =adenosine triphosphate. ND = Not determined, NA = Not applicable, NC =No change. TC50 = concentration that produced a half-maximal response.TC50 values were estimated from the graphs presented in FIGS. 1-46. Ctox= Estimated sustained plasma concentration where toxicity would beexpected to occur in vivo. Blank boxes (line-out) = TC50 not achievedand therefore not determined.

TABLE 2 Total GSH Percent Membrane Caspase 3 Compound TC₅₀ Change inLipid Activity Name/Number (μM) Total GSH Peroxidation (Index/Dose)Summary of Oxidative Stress and Apoptosis (RAT CARDIOMYOCYTES) CLIENTCompound A CM 1 HR >300 −16.2 0 NC CLIENT Compound A CM 3 HR 296 −51.5 0NC CLIENT Compound A CM 6 HR 205 −97.3 0 NC CLIENT Compound A CM 24 HR214 −88.5 0 NC ADRIAMYCIN CM 1 HR >300 −19.3 0 NC ADRIAMYCIN CM 3 HR 61−94.8 0 NC ADRIAMYCIN CM 6 HR 23 −96.2 0 NC ADRIAMYCIN CM 24 HR 1 −86.10 NC IDARUBICIN CM 24 HR <1 −100.0 0 NC MITOXANTRONE CM 24 HR <1 −100.00 NC DAUNORUBICIN CM 24 HR <1 −100.0 0 1/10 PIRARUBICIN CM 24 HR 6 −96.30 NC EPIRUBICIN CM 24 HR 13 −100.0 0 1/20 RITONAVIR CM 24 HR 11 −100.0 0NC EFAVIRENZ CM 24 HR 83 −100.0 2 NC LOPINAVIR CM 24 HR 69 −91.7 0 1/100 DELAVIRDINE CM 24 HR >300 −48.5 0  1/100 ABACAVIR CM 24 HR >300−37.7 2 NC INDINAVIR CM 24 HR 71 −89.2 1 NC NEVIRAPINE CM 24 HR 251−58.6 1 NC AZT CM 24 HR >300 NC 0 NC ROTENONE CM 24 HR 7 −80.8 3 NCCAMPTOTHECIN CM 24 HR >300 −31.5 0  6/300 Summary of Oxidative Stressand Apoptosis (H4IIE CELLS) CLIENT Compound A H4IIE 24 HR 208 −85.8 0 NCADRIAMYCIN H4IIE 24 HR 28 −96.0 0 2/20 IDARUBICIN H4IIE 24 HR 4 −100.0 010/5  DAUNORUBICIN H4IIE 24 HR 16 −99.3 0 8/10 PIRARUBICIN H4IIE 24 HR17 −1.5 0 8/10 DOXORUBICIN H4IIE 24 HR 48 −93.6 1 8/50 EPIRUBICIN H4IIE24 HR 52 −90.6 0 8/50 MITOXANTRONE H4IIE 24 HR 247 −58.1 0 4/10EFAVIRENZ H4IIE 24 HR 170 −99.2 0 NC RITONAVIR H4IIE 24 HR 92 −79.7 0 1/300 DELAVIRDINE H4IIE 24 HR 96 −73.9 1 NC LOPINAVIR H4IIE 24 HR 99−70.3 0  1/300 ABACAVIR H4IIE 24 HR 213 −63.1 0 NC INDINAVIR H4IIE 24 HR249 −57.1 1 NC NEVIRAPINE H4IIE 24 HR >300 NC 1 NC AZT H4IIE 24 HR >300NC 0 NC ROTENONE H4IIE 24 HR 0.05 −100.0 2  1/100 CAMPTOTHECIN H4IIE 24HR 3 −76.9 1  5/100 CM = Rat Cardiomyocyte. GSH Data: Decrease in TotalGSH indicated by (−). Increase in Total GSH indicated by (+). MembraneLipid Peroxidation Data: 0 = No Change, 1 = Modest increase with maximumvalues ≦15 pg/mL, 2 = Concentration related increase with maximum values≧15 pg/mL, 3 = Concentration related increase with a maximum value ≧30pg/mL. Caspase 3 Data: 0-200 = 1, 200-400 = 2, 400-600 = 3, 600-800 = 4,800-1000 = 5, 1000-1200 = 6, 1200-1400 = 7, 1400-1600 = 8, 1600-1800 =9, 1800-2000 = 10. ND = Not determined, NA = Not applicable, NC = Nochange. TC50 = concentration that produced a half-maximal response. TC50values were estimated from the graphs presented in FIGS. 1-46.

TABLE 3 Highest Soluble Compound Concentration Name/Number Tested (μM)Solubility Summary of Test Compounds in Culture Medium (RATCARDIOMYOCYTES) CLIENT Compound A CM 1 HR 300 CLIENT Compound A CM 3 HR300 CLIENT Compound A CM 6 HR 300 CLIENT Compound A CM 24 HR 300ADRIAMYCIN CM 1 HR 100 ADRIAMYCIN CM 3 HR 100 ADRIAMYCIN CM 6 HR 100ADRIAMYCIN CM 24 HR 100 IDARUBICIN CM 24 HR 20 MITOXANTRONE CM 24 HR 300DAUNORUBICIN CM 24 HR 100 PIRARUBICIN CM 24 HR 50 EPIRUBICIN CM 24 HR300 RITONAVIR CM 24 HR 100 EFAVIRENZ CM 24 HR 300 LOPINAVIR CM 24 HR 50DELAVIRDINE CM 24 HR 300 ABACAVIR CM 24 HR 300 INDINAVIR CM 24 HR 300NEVIRAPINE CM 24 HR 300 AZT CM 24 HR 300 ROTENONE CM 24 HR 100CAMPTOTHECIN CM 24 HR 10 Solubility Summary of Test Compounds in CultureMedium (H4IIE CELLS) CLIENT Compound A H4IIE 24 HR 300 ADRIAMYCIN H4IIE24 HR 100 IDARUBICIN H4IIE 24 HR 20 DAUNORUBICIN H4IIE 24 HR 100PIRARUBICIN H4IIE 24 HR 50 DOXORUBICIN H4IIE 24 HR 300 EPIRUBICIN H4IIE24 HR 100 MITOXANTRONE H4IIE 24 HR 300 EFAVIRENZ H4IIE 24 HR 300RITONAVIR H4IIE 24 HR 100 DELAVIRDINE H4IIE 24 HR 300 LOPINAVIR H4IIE 24HR 50 ABACAVIR H4IIE 24 HR 300 INDINAVIR H4IIE 24 HR 300 NEVIRAPINEH4IIE 24 HR 300 AZT H4IIE 24 HR 300 ROTENONE H4IIE 24 HR 100CAMPTOTHECIN H4IIE 24 HR 10 CM = Rat Cardiomyocyte. Note: The solubilityvalues above represent the highest concentration tested at which thecompound remained soluble. Solubility was assessed at 37° C. in thedosing medium, which contained 20% serum.

TABLE 4 Summary of P-glycoprotein (PgP) Binding (H4IIE CELLS) The H4IIEcells possess high levels of PgP protein in the outer membrane. As aresult compounds submitted for toxicity evaluations are also evaluatedfor their potential binding to PgP. Cells are incubated with and withoutcyclosporin A (CSA) (a PgP inhibitor) at a single exposure concentration(50 μM) and the difference in toxicity determined with the MTT assay.Compounds with increased toxicity in the presence of CSA have a highprobability of binding to PgP proteins. Compound % Control % Control %Name/Number (Compound) (Compound + CSA) Difference CLIENT Compound AH4IIE 24 HR 119.2 89.2 25.2 ADRIAMYCIN H4IIE 24 HR 8.0 0.0 ND IDARUBICINH4IIE 24 HR 0.8 0.0 ND DAUNORUBICIN H4IIE 24 HR 9.3 5.5 ND PIRARUBICINH4IIE 24 HR 51.9 33.6 35.2 DOXORUBICIN H4IIE 24 HR 59.3 13.7 76.9EPIRUBICIN H4IIE 24 HR 59.7 11.8 80.3 MITOXANTRONE H4IIE 24 HR 69.9 10.485.1 EFAVIRENZ H4IIE 24 HR 89.0 70.1 21.3 RITONAVIR H4IIE 24 HR 100.681.5 19.0 DELAVIRDINE H4IIE 24 HR 89.8 62.3 30.7 LOPINAVIR H4IIE 24 HR98.0 82.1 16.2 ABACAVIR H4IIE 24 HR 97.6 104.8 NC INDINAVIR H4IIE 24 HR106.0 91.3 13.9 NEVIRAPINE H4IIE 24 HR 115.5 90.2 21.9 AZT H4IIE 24 HR116.6 99.0 NC ROTENONE H4IIE 24 HR 0.0 0.0 ND CAMPTOTHECIN H4IIE 24 HR23.6 26.0 ND ND = Not determined (% control values <70% for compound aretypically not determined because of high cytotoxicity). NA = Notapplicable. NC = No change. PgP Interaction Ranking (based on %Difference): 1-20% = Low interaction, 20-50% = Moderate interaction,50-100% High interaction.

TABLE 5 Summary of Metabolic Activation % Glutathione % GlutathioneCompound Remaining Rat Remaining Dog Name/Number Microsomes MicrosomesClient Compounds COMP A 66.9 84.1 COMP B 58.5 47.4 COMP C 69.3 76.9 2064.9 59.3 COMP C 46.3 67.8 COMP D 6.7 33.0 31 72.6 75.4 39 0.0 11.6 COMPE 96.6 76.9 41 84.9 84.4 Controls APAP 100 μM 74.0 83.7 APAP 1000 μM65.9 66.0 APAP = Acetaminophen. NOTE: Client compounds were analyzed at100 μM.

TABLE 6 Summary of Metabolic Stability % Parent % Parent 1 μM CompoundRemaining Rat Remaining Dog Name/Number Microsomes Microsomes COMP A82.0 63.5 COMP B 52.0 51.5 COMP C 13.5 56.5 COMP D 52.5 6.5 COMP E 33.589.5 MIDAZOLAM 0.0 0.0

TABLE 7 Summary of General Cytotoxicity: Client Compounds (48 HRExposure) Ctox Ranking (μM) - Probability of in vivo Effects 1 High 2021 Moderate 50 51 Low 300 Cell Number MemTox MTT ATP Predicted CompoundTC₅₀ TC₅₀ TC₅₀ TC₅₀ C_(tox) Name/Number (μM) (μM) (μM) (μM) (μM) COMP BRAT PRIMARY 10 79 8 7 ND HEPATOCYTE 48 HR COMP D RATPRIMARY >300 >300 >300 >300 ND HEPATOCYTE 48 HR COMP B DOG PRIMARY 8 6 87 ND HEPATOCYTE 48 HR COMP D DOG PRIMARY >300 >300 >300 >300 NDHEPATOCYTE 48 HR MemTox = Membrane permeability: AK = Adenylate kinase,GST = α-glutathione S-transferase (membrane leakage). MTT =3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, ATP =adenosine triphosphate. ND = Not determined, NA = Not applicable, NC =No change. TC50 = concentration that produced a half-maximal response.TC50 values were estimated from the graphs presented in FIGS. 1-4. Ctox= Estimated sustained plasma concentration where toxicity would beexpected to occur in vivo. Blank boxes (line-out) = TC50 not achievedand therefore not determined.

TABLE 8 Summary of Oxidative Stress and Apoptosis Total GSH PercentMembrane Caspase 3 Compound TC₅₀ Change in Lipid Activity Name/Number(μM) Total GSH Peroxidation (Index/Dose) COMP B RAT PRIMARY 8 −71.4 0 NCHEPATOCYTE 48 HR COMP D RAT PRIMARY >300 −38.9 0 NC HEPATOCYTE 48 HRCOMP B DOG PRIMARY 8 −91.0 3 NC HEPATOCYTE 48 HR COMP D DOG PRIMARY >300NC 0 NC HEPATOCYTE 48 HR GSH Data: Decrease in Total GSH indicated by(−). Increase in Total GSH indicated by (+). Membrane Lipid PeroxidationData: 0 = No Change, 1 = Modest increase with maximum values ≦15 pg/mL,2 = Concentration related increase with maximum values ≧15 pg/mL, 3 =Concentration related increase with a maximum value ≧30 pg/mL. Caspase 3Data: 0-200 = 1, 200-400 = 2, 400-600 = 3, 600-800 = 4, 800-1000 = 5,1000-1200 = 6, 1200-1400 = 7, 1400-1600 = 8, 1600-1800 = 9, 1800-2000 =10. ND = Not determined, NA = Not applicable, NC = No change. TC50 =concentration that produced a half-maximal response. TC50 values wereestimated from the graphs presented in FIGS. 1-4.

TABLE 9 Solubility Summary of Test Compounds in Culture Medium HighestSoluble Compound Concentration Name/Number Tested (μM) COMP B RATPRIMARY 20-50 (a) HEPATOCYTE 48 HR COMP D RAT PRIMARY 300 HEPATOCYTE 48HR COMP B DOG PRIMARY 20-50 (a) HEPATOCYTE 48 HR COMP D DOG PRIMARY 300HEPATOCYTE 48 HR Note: The solubility values above represent the highestconcentration tested at which the compound remained soluble. Solubilitywas assessed at 37° C. in the dosing medium, which contained 20% serum.Compound COMP B was soluble up to and including 20 μM at both 0 hr and48 hr reads. The compound was borderline soluble (3X background) at 50μM at both 0 hr and 48 hr reads.

TABLE 10A Non-GLP In Vitro Toxicity Screening Results 24 hr ExposureAlamar Batch Cell GST MTT Blue ATP Predicted Compound Number or NumberTC₅₀ TC₅₀ TC₅₀ TC₅₀ C_(tox) Number Lot Number TC₅₀ (Dil.) (Dil.) (Dil.)(Dil.) (Dil.) (Dil.) COMP A NA 0.0016 0.0016 0.0016 ND 0.0014 0.001(1:625) (1:625) (1:625) (1:714) (1:1000)

TABLE 10B Non-GLP In Vitro Toxicity Screening Results: 6 Hr and 24 HrExposures Cell Alamar Batch Number GST MTT Blue ATP Predicted CompoundNumber or TC₅₀ TC₅₀ TC₅₀ TC₅₀ TC₅₀ C_(tox) Number Lot Number (Dil.)(Dil.) (Dil.) (Dil.) (Dil.) (Dil.) Comp A Rat NA 0.009 0.006 0.004 ND0.006 ND Primary 6 Hr (1:111) (1:167) (1:250) (1:167) Comp A Rat NA0.007 0.006 0.004 ND 0.002 ND Primary 24 Hr (1:143) (1:167) (1:250)(1:500) Cell Alamar Batch Number GST MTT Blue ATP Predicted CompoundNumber or TC₅₀ TC₅₀ TC₅₀ TC₅₀ TC₅₀ C_(tox) Number Lot Number (pH) (pH)(pH) (pH) (pH) (pH) pH Test Rat NA ND ND 6.2 ND 6.2 ND Primary 6 Hr pHTest Rat NA ND ND 6.2 ND 6.4 ND Primary 24 Hr

TABLE 10C Non-GLP In Vitro Toxicity Screening Results (24 Hr Exposure)Batch Cell Mem Number Number Tox MTT ATP Compound or Lot TC₅₀ TC₅₀ TC₅₀TC₅₀ Predicted Number Number (Dil.) (Dil.) (Dil.) (Dil.) C_(tox) (Dil.)COMP A NA 0.049 0.060 0.040 0.014 0.009 NRK 6 HR (1:20) (1:17) (1:25)(1:71) (1:111) COMP A NA 0.057 0.044 0.012 0.006 0.009 NRK 24 HR (1:18)(1:23) (1:83) (1:167) (1:111) Batch Cell Mem Number Number Tox MTT ATPCompound or Lot TC₅₀ TC₅₀ TC₅₀ TC₅₀ Predicted Number Number (pH) (pH)(pH.) (pH) C_(tox) (pH.) PH TEST NA ND 6.2 6.2 6.2 6.8 NRK 6 HR PH TESTNA ND 6.2 6.2 6.2 6.8 NRK 24 HR Batch Cell Number Number GST MTT ATPCompound or Lot TC₅₀ TC₅₀ TC₅₀ TC₅₀ Predicted Number Number (μM) (μM)(μM) (μM) C_(tox) (μM) CAMPTO- NA ND 300 300 300 0.1 THECIN NRK 6 HRCAMPTO- NA ND 100 100 9 0.1 THECIN NRK 24 HR ROTENONE NA ND 10 10 10 1NRK 6 HR ROTENONE NA ND 10 10 10 1 NRK 24 HR For Tables 10A-C: GST =α-glutathione S-transferase (membrane permeability). MTT =3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. ATP =adenosine triphosphate. ND = Not determined. NA = Not applicable. NC =No change. Dil = Dilution. TC50 = concentration that produced ahalf-maximal response. TC50 values were estimated from the graphspresented in FIG. 1. Ctox = Estimated sustained plasma concentrationwhere toxicity would be expected to occur in vivo. * = Ctox was based onthe responses observed for two indicators of mitochondrial health. Thetrue Ctox may be higher. Exposure Concentrations (Dilutions): 2000 Dil.= 0.0005 1000 Dil. = 0.001 500 Dil. = 0.002 100 Dil. = 0.01 50 Dil. =0.02 10 Dil. = 0.1 1 Dil. = 0.5

TABLE 11A Non-GLP In Vitro Toxicity Screening Results For The OxidativeStress-Cluster Batch Total GSH Percent Membrane Caspase 3 CompoundNumber or TC50 Change in Lipid Activity Number Lot Number (Dil.) TotalGSH Peroxidation Index/Dose COMP A NA 0.0015 100.0 1 3/0.002 (1:667)(1:500)

TABLE 11B Non-GLP In Vitro Toxicity Screening Results For The OxidativeStress and Apoptosis Clusters: 6 Hr and 24 Hr Exposures Caspase 3 BatchTotal GSH Percent Membrane Activity Compound Number or TC50 Change inLipid Index/Dose Number Lot Number (Dil.) Total GSH Peroxidation (Dil.)Comp A Rat NA 0.002 −81.9 3 1/0.001 Primary 6 Hr (1:500) (1:1000) Comp ARat NA 0.001 −73.3 3 1/0.002 Primary 24 Hr (1:1000) (1:500) Caspase 3Batch Total GSH Percent Membrane Activity Compound Number or TC50 Changein Lipid Index/Dose Number Lot Number (μM) Total GSH Peroxidation (pH)pH Test Rat NA ND ND ND 2/6.8  Primary 6 Hr pH Test Rat NA ND ND ND1/6.8  Primary 24 Hr

TABLE 11C Non-GLP In Vitro Toxicity Screening Results For The OxidativeStress and Apoptosis Panels: 6 Hr and 24 Hr Exposures Caspase 3 BatchTotal GSH Percent Membrane Activity Compound Number or TC50 Change inLipid Index/Dose Number Lot Number (Dil.) Total GSH Peroxidation (Dil.)COMP A NA 0 100.0 1 NC NRK 6 HR COMP A NA 0 100.0 3   2/0.01 NRK 24 HRPercent Caspase 3 Batch Total GSH Change Membrane Activity CompoundNumber or TC50 in Total Lipid Index/Dose Number Lot Number (pH) GSHPeroxidation (pH) PH TEST NA ND ND ND  1/6.0 NRK 6 HR PH TEST NA ND NDND  1/6.0 NRK 24 HR Caspase 3 Batch Total GSH Percent Membrane ActivityCompound Number or TC50 Change in Lipid Index/Dose Number Lot Number(μM) Total GSH Peroxidation (μM) CAMPTOTHECIN NA ND ND ND  3/100 NRK 6HR CAMPTOTHECIN NA ND ND ND 10/300 NRK 24 HR ROTENONE NA ND ND ND NC NRK6 HR ROTENONE NA ND ND ND 2/10 NRK 24 HR

TABLE 12A Solubility of the test compounds in culture media containing20% serum Highest Soluble Compound Concentration Number (Dil.) COMP A0.002 (1:500)

TABLE 12B Solubility of the Test Compounds in Culture Media Containing10% Serum Highest Soluble Compound Concentration Number (Dil.) Comp ARat 0.002 Primary 6 Hr (1:500) Comp A Rat 0.002 Primary 24 Hr (1:500)Highest Soluble Compound Concentration Number (pH) pH Test Rat 6.5Primary 6 Hr pH Test Rat 6.5 Primary 24 Hr

TABLE 12C Solubility of the Test Compounds in Culture Media Containing20% Serum Highest Soluble Compound Concentration Number (Dil.) COMP A0.02 NRK 6 HR COMP A 0.01 NRK 24 HR Highest Soluble CompoundConcentration Number (pH) PH TEST 6.0 NRK 6 HR PH TEST 6.0 NRK 24 HRHighest Soluble Compound Concentration Number (μM) CAMPTOTHECIN 50 NRK 6HR CAMPTOTHECIN 50 NRK 24 HR ROTENONE 10 NRK 6 HR ROTENONE 10 NRK 24 HR

TABLE 13A P-glycoprotein binding The H4IIE cells possess high levels ofPgP protein in the outer membrane. As a result compounds submitted fortoxicity evaluations are also evaluated for their potential binding toPgP. Cells are incubated with and without cyclosporin A (CSA) (a PgPinhibitor) at a single exposure concentration (50 μM) and the differencein toxicity determined with the MTT assay. Compounds with increasedtoxicity in the presence of CSA have a high probability of binding toPgP proteins. Batch % Control Compound Number or % Control Compound + %Number Lot Number Compound CSA Difference COMP A NA 6.6 6.3 3.5

TABLE 14 Summary of General Cytotoxicity Ctox Ranking (μM) - Probabilityof in vivo Toxicity 1  Toxic  20 21  Caution  50 51  LowestToxicity  300 Cell Number MemTox MTT BrdU ATP Predicted Compound TC₅₀TC₅₀ TC₅₀ TC₅₀ TC₅₀ C_(tox) Name/Number (μg/mL) (μg/mL) (μg/mL) (μg/mL)(μg/mL) (μg/mL) COMP A; SK-MEL28 24 HR — — (0.25:0.05)  ND — ND COMP A(LOW); SK-MEL28 24 HR — — — — — (0.6:0.12) COMP A (LOW); SK-MEL28 72 HR(0.1:0.2) — (0.04:0.008) (0.1:0.2) (0.1:0.2) ND COMP A; HUMAN HEP 24 HR— — — ND — ND COMP A; HUVEC 24 HR — — — ND — ND COMP A; C32 24 HR — — —ND — ND COMP A; NHEM 24 HR — — — ND — ND Cell Number MemTox MTT BrdU ATPPredicted Compound TC₅₀ TC₅₀ TC₅₀ TC₅₀ TC₅₀ C_(tox) Name/Number (μM)(μM) (μM) (μM) (μM) (μM) CAMPTOTHECIN SK-MEL28 — — 241 ND 293  0.1 24 HRCAMPTOTHECIN #2 SK-MEL28 — — 249 0.1 — 0.1 24 HR CAMPTOTHECIN #2SK-MEL28   0.1 —    0.1 0.2   0.1 ND 72 HR CAMPTOTHECIN HUMAN HEP — — —ND — 23   24 HR CAMPTOTHECIN HUVEC 85  13  41 ND 40  0.1 24 HRCAMPTOTHECIN — — 213 ND — 0.1 C32 24 HR CAMPTOTHECIN — — — ND — 33  NHEM 24 HR ROTENONE; SK-MEL28 24 HR — — — ND — 1   ROTENONE #2; SK-MEL28— — — 9   — 0   24 HR ROTENONE #2; SK-MEL28 1 —    0.2 44   2 ND 72 HRROTENONE; HUMAN HEP 24 HR — —  3 ND 6 0.6 ROTENONE; HUVEC 24 HR — — — ND— 0.1 ROTENONE; C32 24 HR — — — ND — ND ROTENONE; NHEM 24 HR — — — ND —10   MemTox = Membrane permeability: AK = Adenylate kinase, GST =α-glutathione S-transferase (membrane leakage). MTT =3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, ATP =adenosine triphosphate. ND = Not determined, NA = Not applicable, NC =No change. TC50 = concentration that produced a half-maximal response.TC50 values were estimated from the graphs presented in FIGS. 68-87.Ctox = Estimated sustained plasma concentration where toxicity would beexpected to occur in vivo.

TABLE 15 Summary of Oxidative Stress and Apoptosis Total GSH PercentMembrane Caspase 3 Compound TC₅₀ Change in Lipid Activity Name/Number(μg/mL) GSH Peroxidation (Index/Dose) COMP A; SK-MEL28 24 HR — −30.6 01/(12.5:2.5)  COMP A (LOW); SK-MEL28 24 HR — −27.2 0 1/(1.25:0.25) COMPA (LOW); SK-MEL28 72 HR (0.1:0.02) −79.4 0 1/(2.5:0.5)  COMP A; HUMANHEP 24 HR — −23.7 0 NC COMP A; HUVEC 24 HR (156:31)  −53.9 2 NC COMP A;C32 24 HR — NC 0 NC COMP A; NHEM 24 HR — −30.4 1 NC Total GSH PercentMembrane Caspase 3 Compound TC₅₀ Change in Lipid Activity Name/Number(μM) Total GSH Peroxidation (Index/Dose) CAMPTOTHECIN; SK-MEL28 24 HR —−46.0 0 3/300 CAMPTOTHECIN #2; 10 −57.9 0 3/100 SK-MEL28 24 HRCAMPTOTHECIN #2 0.1 −88.8 1 3/100 SK-MEL28 72 HR CAMPTOTHECIN 44 −69.9 13/100 HUMAN HEP 24 HR CAMPTOTHECIN; HUVEC 24 HR 1 −85.8 2 1/300CAMPTOTHECIN; C32 24 HR 1 −41.1 0 4/300 CAMPTOTHECIN; NHEM 24 HR — NC 13/300 ROTENONE; SK-MEL28 24 HR 9 52.8 3 NC ROTENONE #2; SK-MEL28 24 HR 949.9 2 NC ROTENONE #2; SK-MEL28 72 HR 0.1 85.1 3 NC ROTENONE; HUMAN HEP24 HR 1 95.9 0 NC ROTENONE; HUVEC 24 HR 10 11.3 1 NC ROTENONE; C32 24 HR10 12.9 0 NC ROTENONE; NHEM 24 HR — NC 2 NC GSH Data: Decrease in TotalGSH indicated by (−); Increase in Total GSH indicated by (+). MembraneLipid Peroxidation Data: 0 = No Change, 1 = Modest increase with maximumvalues ≦ 15 pg/mL, 2 = Concentration related increase with maximumvalues ≧ 15 pg/mL, 3 = Concentration related increase with a maximumvalue ≧ 30 pg/mL. Caspase 3 Data: 0-200 = 1, 200-400 = 2, 400-600 = 3,600-800 = 4, 800-1000 = 5, 1000-1200 = 6, 1200-1400 = 7, 1400-1600 = 8,1600-1800 = 9, 1800-2000 = 10. ND = Not determined, NA = Not applicable,NC = No change. TC50 = concentration that produced a half-maximalresponse. TC50 values were estimated from the graphs presented in FIGS.1-19. Blank boxes (line-out) = TC50 not achieved and therefore notdetermined.

TABLE 16 Solubility Summary of Test Compounds in Culture Medium HighestSoluble Compound Concentration Name/Number (μg/mL) COMP A; SK-MEL28 24HR (250:50) COMP A (LOW); SK-MEL28 24 HR  (2.5:0.5) COMP A (LOW);SK-MEL28 72 HR  (2.5:0.5) COMP A; HUMAN HEP 24 HR (250:50) COMP A; HUVEC24 HR (250:50) COMP A; C32 24 HR (250:50) COMP A; NHEM 24 HR (250:50)Highest Soluble Compound Concentration Name/Number (μM) CAMPTOTHECIN;SK-MEL28 24 HR 10 CAMPTOTHECIN #2; SK-MEL28 24 HR 10 CAMPTOTHECIN #2;SK-MEL28 72 HR 10 CAMPTOTHECIN; HUMAN HEP 24 HR 10 CAMPTOTHECIN; HUVEC24 HR 10 CAMPTOTHECIN; C32 24 HR 10 CAMPTOTHECIN; NHEM 24 HR 10ROTENONE; SK-MEL28 24 HR 10 ROTENONE #2; SK-MEL28 24 HR 100* ROTENONE#2; SK-MEL28 72 HR 100* ROTENONE; HUMAN HEP 24 HR 10 ROTENONE; HUVEC 24HR 10 ROTENONE; C32 24 HR 10 ROTENONE; NHEM 24 HR 100* Note: Thesolubility values above represent the highest concentration at which thecompound remained soluble. Solubility was assessed at 37° C. in thedosing medium, which contained 20% serum. Red highlight indicates lowsolubility. Note: Rotenone exposures for SK-MEL28 (24, 72 hr) and NHEM(24 hr) were done up to and including 100 μM. All other Rotenoneexposures in this group were done up to and including 10 μM.

TABLE 17 Summary of P-glycoprotein (PgP) Binding Compound % Control %Control % Name/Number (Compound) (Compound + CSA) Difference COMP A;SK-MEL28 24 HR 48.2 42.3 12.3 COMP A (LOW); SK-MEL28 24 HR ND ND ND COMPA (LOW); SK-MEL28 72 HR ND ND ND COMP A; HUMAN HEP 24 HR 113.5  101.1 NC COMP A; HUVEC 24 HR 70.5 88.3 NC COMP A; C32 24 HR ND ND ND COMP A;NHEM 24 HR ND ND ND CAMPTOTHECIN; SK-MEL28 24 HR 56.5 63.3 NCCAMPTOTHECIN #2; SK-MEL28 24 HR ND ND ND CAMPTOTHECIN #2; SK-MEL28 72 HRND ND ND CAMPTOTHECIN; HUMAN HEP 24 HR 88.5 65.8 25.7 CAMPTOTHECIN;HUVEC 24 HR 29.4 18.0 ND CAMPTOTHECIN; C32 24 HR ND ND ND CAMPTOTHECIN;NHEM 24 HR ND ND ND ROTENONE; SK-MEL28 24 HR 98.2 117.3  NC ROTENONE #2;SK-MEL28 24 HR ND ND ND ROTENONE #2; SK-MEL28 72 HR ND ND ND ROTENONE;HUMAN HEP 24 HR 97.1 60.9 37.2 ROTENONE; HUVEC 24 HR 75.4 77.8 NCROTENONE; C32 24 HR ND ND ND ROTENONE; NHEM 24 HR ND ND ND The H4IIEcells possess high levels of PgP protein in the outer membrane. As aresult compounds submitted for toxicity evaluations are also evaluatedfor their potential binding to PgP. Cells are incubated with and withoutcyclosporin A (CSA) (a PgP inhibitor) at a single exposure concentration(50 μM) and the difference in toxicity determined with the MTT assay.Compounds with increased toxicity in the presence of CSA have a highprobability of binding to PgP proteins. ND = Not determined (% controlvalues <70% for compound are typically not determined because of highcytotoxicity). NA = Not applicable. NC = No change. PgP InteractionRanking (based on % Difference): 1-20% = Low interaction, 20-50% =Moderate interaction, 50-100% High interaction.

1. A method of determining a level of toxicity in normal tissue for ananti-tumor compound, the method comprising the steps of: (a) culturingat least one first cell type in the presence of a plurality ofconcentrations of an anti-tumor compound, wherein the first cell type isa proliferating cell derived from a tumor; (b) culturing at least onesecond cell type in the presence of a plurality of concentrations of theanti-tumor compound, wherein the second cell type is a cell derived froma normal tissue of the same species and tissue as the first cell type;(c) culturing at least one third cell type in the presence of aplurality of concentrations of the anti-tumor compound, wherein thethird cell type is derived from a normal tissue of the same species butdifferent tissue as the first and second cell types; (d) measuring atleast one indicator of cellular replication, at least one indicator ofmitochondrial function, and at least one indicator of cell viability atfour or more concentrations of the anti-tumor compound for each of theat least three cell types; (e) determining a potency/efficacy of theanti-tumor compound based on the measurements of the at least oneindicator of cellular replication in the first cell type; (f)determining a specificity of the anti-tumor compound by comparing atleast one of the measurements from (d) for at least one of theindicator(s) of cellular replication, mitochondrial function, and cellviability for the first cell type to at least one of the measurementsfrom (d) for at least one of the indicator(s) of cellular replication,mitochondrial function, and cell viability for the second cell type; and(g) comparing the results obtained for at least one of the indicator(s)of cellular replication, mitochondrial function, and cell viability forthe at least one third cell type to the results obtained for the sameindicator(s) for the first and second cell types.
 2. The method of claim1, further comprising determining a concentration of the anti-tumorcompound that produces a half maximal toxic effect (TC₅₀) for each ofthe indicator of cellular replication, indicator of mitochondrialfunction, and indicator of cell viability for each of the first andsecond cell types.
 3. The method of claim 2, further comprisingcomparing the TC₅₀ for the indicator of cellular replication in thefirst cell type to the TC₅₀ for at least one of the indicators ofmitochondrial function and cell viability for the second cell type. 4.The method of claim 1, wherein the plurality of concentrations of theanti-tumor compound are selected from a concentration range from 0micromolar and to about 300 micromolar.
 5. The method of claim 1,further comprising plotting the measurements for each cell healthindicator on a graph as a function of concentration for each cell healthindicators of the anti-tumor compound.
 6. The method of claim 1, whereinthe measurements of each of the cell health indicators are expressedrelative to a control measurement as a function of concentration of theanti-tumor compound.
 7. The method of claim 6, wherein the measurementsof all of the cell health indicators are plotted on a single graph. 8.The method of claim 1, further comprising measuring at least oneadditional indicator of cell health selected from the group consistingof indicators of intracellular energy balance, indicators of cellmembrane integrity, indicators of cell mortality, indicators ofoxidative stress, indicators of metabolic stability, indicators ofmetabolic activation, indicators of enzyme induction, indicators ofenzyme inhibition, and indicators of interaction with cell membranetransporters.
 9. The method of claim 1, further comprising the steps of:determining a relative abundance of the anti-tumor compound's intendedtarget in each of the at least three cell types; and normalizing thetoxicity response to the target in each cell type. 10-20. (canceled)